M.G. Myriam Hunink, G. Scott Gazelle
Ronald G. Blasberg, Juri Gelovani Tjuvajev
John R. Forder, Gerald M. Pohost
It has been proposed — but remains controversial — that estrogen’s effects on various tissues may be mediated by different cell signaling pathways. Researchers have identified a synthetic ligand that activates only a subset of these pathways, suggesting that bypass of the traditional estrogen pathway can prevent bone loss without associated side effects on reproductive organs.
Antimicrobial peptides are effector molecules of innate immunity with microbicidal and pro- or anti-inflammatory activities. Their role is now widening following evidence that one such multifunctional peptide, LL-37, induces angiogenesis, a process essential for host defense, wound healing, and tissue repair.
The presence in newborns of a mature and functional CD8+ T cell response to congenital cytomegalovirus infection suggests that the machinery necessary to prime such responses is present in utero and raises questions related to neonatal vaccination.
Tissue tropism of clinical ocular and genital Chlamydia trachomatis strains is shown to be linked to the tryptophan synthase genotype. It is suggested that, in the presence of IFN-γ, which depletes available tryptophan, there exist unique host-parasite interactions that may contribute to persistent chlamydial infection.
Richard P. Morrison
It has been found that 4-estren-3α,17β-diol, a synthetic ligand for the estrogen receptor (ER) or androgen receptor (AR), which does not affect classical transcription, reverses bone loss in ovariectomized females or orchidectomized males without affecting the uterus or seminal vesicles, demonstrating that the classical genotropic actions of sex steroid receptors are dispensable for their bone-protective effects, but indispensable for their effects on reproductive organs. We have now investigated the mechanism of action of this compound. We report that, identically to 17β-estradiol or dihydrotestosterone, but differently from raloxifene, estren alters the activity of Elk-1, CCAAT enhancer binding protein–β (C/EBPβ), and cyclic adenosine monophosphate–response element binding protein (CREB), or c-Jun/c-Fos by an extranuclear action of the ER or AR, resulting in activation of the Src/Shc/ERK pathway or downregulation of JNK, respectively. All of these effects are non–sex specific, require only the ligand-binding domain of the receptor, and are indispensable for the antiapoptotic action of these ligands on osteoblastic and HeLa cells. Moreover, administration of 17β-estradiol or 4-estren-3α,17β-diol to ovariectomized mice induces phosphorylation of ERKs, Elk-1, and C/EBPβ, downregulates c-Jun, and upregulates the expression of egr-1, an ERK/SRE target gene. Kinase-initiated regulation of commonly used transcription factors offers a molecular explanation for the profound skeletal effects of sex steroid receptor ligands, including synthetic ones that are devoid of classical transcriptional activity.
Stavroula Kousteni, Li Han, Jin-Ran Chen, Maria Almeida, Lilian I. Plotkin, Teresita Bellido, Stavros C. Manolagas
Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor–like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.
Rembert Koczulla, Georges von Degenfeld, Christian Kupatt, Florian Krötz, Stefan Zahler, Torsten Gloe, Katja Issbrücker, Pia Unterberger, Mohamed Zaiou, Corinna Lebherz, Alexander Karl, Philip Raake, Achim Pfosser, Peter Boekstegers, Ulrich Welsch, Pieter S. Hiemstra, Claus Vogelmeier, Richard L. Gallo, Matthias Clauss, Robert Bals
The present study evaluates the potential of third-generation lentivirus vectors with respect to their use as in vivo–administered T cell vaccines. We demonstrate that lentivector injection into the footpad of mice transduces DCs that appear in the draining lymph node and in the spleen. In addition, a lentivector vaccine bearing a T cell antigen induced very strong systemic antigen-specific cytotoxic T lymphocyte (CTL) responses in mice. Comparative vaccination performed in two different antigen models demonstrated that in vivo administration of lentivector was superior to transfer of transduced DCs or peptide/adjuvant vaccination in terms of both amplitude and longevity of the CTL response. Our data suggest that a decisive factor for efficient T cell priming by lentivector might be the targeting of DCs in situ and their subsequent migration to secondary lymphoid organs. The combination of performance, ease of application, and absence of pre-existing immunity in humans make lentivector-based vaccines an attractive candidate for cancer immunotherapy.
Christoph Esslinger, Laurence Chapatte, Daniela Finke, Isabelle Miconnet, Philippe Guillaume, Frédéric Lévy, H. Robson MacDonald
Dominantly inherited mutations in ACTN4, which encodes α-actinin-4, cause a form of human focal and segmental glomerulosclerosis (FSGS). By homologous recombination in ES cells, we developed a mouse model deficient in Actn4. Mice homozygous for the targeted allele have no detectable α-actinin-4 protein expression. The number of homozygous mice observed was lower than expected under mendelian inheritance. Surviving mice homozygous for the targeted allele show progressive proteinuria, glomerular disease, and typically death by several months of age. Light microscopic analysis shows extensive glomerular disease and proteinaceous casts. Electron microscopic examination shows focal areas of podocyte foot-process effacement in young mice, and diffuse effacement and globally disrupted podocyte morphology in older mice. Despite the widespread distribution of α-actinin-4, histologic examination of mice showed abnormalities only in the kidneys. In contrast to the dominantly inherited human form of ACTN4-associated FSGS, here we show that the absence of α-actinin-4 causes a recessive form of disease in mice. Cell motility, as measured by lymphocyte chemotaxis assays, was increased in the absence of α-actinin-4. We conclude that α-actinin-4 is required for normal glomerular function. We further conclude that the nonsarcomeric forms of α-actinin (α-actinin-1 and α-actinin-4) are not functionally redundant. In addition, these genetic studies demonstrate that the nonsarcomeric α-actinin-4 is involved in the regulation of cell movement.
Claudine H. Kos, Tu Cam Le, Sumita Sinha, Joel M. Henderson, Sung Han Kim, Hikaru Sugimoto, Raghu Kalluri, Robert E. Gerszten, Martin R. Pollak
The prostate gland is a rich source of α1-adrenergic receptors (α1-ARs). α1-AR antagonists are commonly used in the treatment of benign prostatic hyperplasia symptoms, due to their action on smooth muscle cells. However, virtually nothing is known about the role of α1-ARs in epithelial cells. Here, by using two human prostate cancer epithelial (hPCE) cell models — primary cells from resection specimens (primary hPCE cells) and an LNCaP (lymph node carcinoma of the prostate) cell line — we identify an α1A subtype of adrenergic receptor (α1A-AR) and show its functional coupling to plasmalemmal cationic channels via direct diacylglycerol (DAG) gating. In both cell types, agonist-mediated stimulation of α1A-ARs and DAG analogues activated similar cationic membrane currents and Ca2+ influx. These currents were sensitive to the α1A-AR antagonists, prazosin and WB4101, and to transient receptor potential (TRP) channel blockers, 2–aminophenyl borate and SK&F 96365. Chronic activation of α1A-ARs enhanced LNCaP cell proliferation, which could be antagonized by α1A-AR and TRP inhibitors. Collectively, our results suggest that α1-ARs play a role in promoting hPCE cell proliferation via TRP channels.
Stephanie Thebault, Morad Roudbaraki, Vadim Sydorenko, Yaroslav Shuba, Loic Lemonnier, Christian Slomianny, Etienne Dewailly, Jean-Louis Bonnal, Brigitte Mauroy, Roman Skryma, Natalia Prevarskaya
Through a combination of fluorescence microscopy and patch-clamp analysis we have identified a striking alteration in K+ channel expression in terminally differentiated human CCR7–CD45RA– effector memory T lymphocytes (TEM). Following activation, TEM cells expressed significantly higher levels of the voltage-gated K+ channel Kv1.3 and lower levels of the calcium-activated K+ channel IKCa1 than naive and central memory T cells (TCM). Upon repeated in vitro antigenic stimulation, naive cells differentiated into Kv1.3highIKCa1low TEM cells, and the potent Kv1.3-blocking sea anemone Stichodactyla helianthus peptide (ShK) suppressed proliferation of TEM cells without affecting naive or TCM lymphocytes. Thus, the Kv1.3highIKCa1low phenotype is a functional marker of activated TEM lymphocytes. Activated myelin-reactive T cells from patients with MS exhibited the Kv1.3highIKCa1low TEM phenotype, suggesting that they have undergone repeated stimulation during the course of disease; these cells may contribute to disease pathogenesis due to their ability to home to inflamed tissues and exhibit immediate effector function. The Kv1.3highIKCa1low phenotype was not seen in glutamic acid decarboxylase, insulin-peptide or ovalbumin-specific and mitogen-activated T cells from MS patients, or in myelin-specific T cells from healthy controls. Selective targeting of Kv1.3 in TEM cells may therefore hold therapeutic promise for MS and other T cell–mediated autoimmune diseases.
Heike Wulff, Peter A. Calabresi, Rameeza Allie, Sung Yun, Michael Pennington, Christine Beeton, K. George Chandy
Recent studies have identified the white adipose tissue (WAT) as an important endocrine organ that regulates energy and glucose metabolism via a number of secreted factors. Mice lacking acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in mammalian triglyceride synthesis, are protected against diet-induced obesity and glucose intolerance because of increased energy expenditure and enhanced insulin sensitivity. Because DGAT1 is highly expressed in WAT, we hypothesized that DGAT1 deficiency affects the expression of adipocyte-derived factors that regulate energy and glucose metabolism. Here we show that the transplantation of DGAT1-deficient WAT decreases adiposity and enhances glucose disposal in wild-type mice. Analysis of DGAT1-deficient WAT revealed a twofold increase in the expression of adiponectin, a molecule that enhances fatty acid oxidation and insulin sensitivity, and this increase may account in part for the transplantation-induced metabolic changes. Our results highlight the importance of the endocrine function of WAT and suggest that an alteration in this function contributes to the increased energy expenditure and insulin sensitivity in DGAT1-deficient mice.
Hubert C. Chen, Dalan R. Jensen, Heather M. Myers, Robert H. Eckel, Robert V. Farese Jr.
The secretory factor VEGF-C has been directly implicated in various physiological processes during embryogenesis and human cancers. However, the importance of the conversion of its precursor proVEGF-C to mature VEGF-C in tumorigenesis, and vessel formation and the identity of the protease(s) that regulate these processes is/are not known. The intracellular processing of proVEGF-C that occurs within the dibasic motif HSIIRR227SL suggests the involvement of the proprotein convertases (PCs) in this process. In addition, furin and VEGF-C were found to be coordinately expressed in adult mouse tissues. Cotransfection of the furin-deficient colon carcinoma cell line LoVo with proVEGF-C and different PC members revealed that furin, PC5, and PC7 are candidate VEGF-C convertases. This finding is consistent with the in vitro digestions of an internally quenched synthetic fluorogenic peptide mimicking the cleavage site of proVEGF-C (220Q-VHSIIRR↓SLP230). The processing of proVEGF-C is blocked by the inhibitory prosegments of furin, PC5, and PACE4, as well as by furin-motif variants of α2-macroglobulin and α1-antitrypsin. Subcutaneous injection of CHO cells stably expressing VEGF-C into nude mice enhanced angiogenesis and lymphangiogenesis, but not tumor growth. In contrast, expression of proVEGF-C obtained following mutation of the cleavage site (HSIIRR227SL to HSIISS227SL) inhibits angiogenesis and lymphangiogenesis as well as tumor growth. Our findings demonstrate the processing of proVEGF-C by PCs and highlight the potential use of PC inhibitors as agents for inhibiting malignancies induced by VEGF-C.
Geraldine Siegfried, Ajoy Basak, James A. Cromlish, Suzanne Benjannet, Jadwiga Marcinkiewicz, Michel Chrétien, Nabil G. Seidah, Abdel-Majid Khatib
Thyroid function depends on processing of the prohormone thyroglobulin by sequential proteolytic events. From in vitro analysis it is known that cysteine proteinases mediate proteolytic processing of thyroglobulin. Here, we have analyzed mice with deficiencies in cathepsins B, K, L, B and K, or K and L in order to investigate which of the cysteine proteinases is most important for proteolytic processing of thyroglobulin in vivo. Immunolabeling demonstrated a rearrangement of the endocytic system and a redistribution of extracellularly located enzymes in thyroids of cathepsin-deficient mice. Cathepsin L was upregulated in thyroids of cathepsin K–/– or B–/–/K–/– mice, suggesting a compensation of cathepsin L for cathepsin K deficiency. Impaired proteolysis resulted in the persistence of thyroglobulin in the thyroids of mice with deficiencies in cathepsin B or L. The typical multilayered appearance of extracellularly stored thyroglobulin was retained in cathepsin K–/– mice only. These results suggest that cathepsins B and L are involved in the solubilization of thyroglobulin from its covalently cross-linked storage form. Cathepsin K–/–/L–/– mice had significantly reduced levels of free thyroxine, indicating that utilization of luminal thyroglobulin for thyroxine liberation is mediated by a combinatory action of cathepsins K and L.
Bianca Friedrichs, Carmen Tepel, Thomas Reinheckel, Jan Deussing, Kurt von Figura, Volker Herzog, Christoph Peters, Paul Saftig, Klaudia Brix
Immunization of newborns against viral infections may be hampered by ineffective CD8+ T cell responses. To characterize the function of CD8+ T lymphocytes in early life, we studied newborns with congenital human cytomegalovirus (HCMV) infection. We demonstrate that HCMV infection in utero leads to the expansion and the differentiation of mature HCMV-specific CD8+ T cells, which have similar characteristics to those detected in adults. High frequencies of HCMV-specific CD8+ T cells were detected by ex vivo tetramer staining as early as after 28 weeks of gestation. During the acute phase of infection, these cells had an early differentiation phenotype (CD28–CD27+CD45RO+, perforinlow), and they acquired a late differentiation phenotype (CD28–CD27-CD45RA+, perforinhigh) during the course of the infection. The differentiated cells showed potent perforin-dependent cytolytic activity and produced antiviral cytokines. The finding of a mature and functional CD8+ T cell response to HCMV suggests that the machinery required to prime such responses is in place during fetal life and could be used to immunize newborns against viral pathogens.
Arnaud Marchant, Victor Appay, Marianne van der Sande, Nicolas Dulphy, Corinne Liesnard, Michael Kidd, Steve Kaye, Olubukola Ojuola, Geraldine M.A. Gillespie, Ana L. Vargas Cuero, Vincenzo Cerundolo, Margaret Callan, Keith P.W.J. McAdam, Sarah L. Rowland-Jones, Catherine Donner, Andrew J. McMichael, Hilton Whittle
We previously reported that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogen’s tryptophan synthase (trpBA) genes. Here, we have applied functional genomics to extend this work and find that the paradigm established for reference serovars also applies to clinical isolates — specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme. Moreover, functional enzyme activity was directly correlated to IFN-γ resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We speculate that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-γ–mediated eradication and thus establish persistent infection.
Harlan D. Caldwell, Heidi Wood, Debbie Crane, Robin Bailey, Robert B. Jones, David Mabey, Ian Maclean, Zeena Mohammed, Rosanna Peeling, Christine Roshick, Julius Schachter, Anthony W. Solomon, Walter E. Stamm, Robert J. Suchland, Lacey Taylor, Sheila K. West, Tom C. Quinn, Robert J. Belland, Grant McClarty
We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic β subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor–1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.
I.R. Garrett, D. Chen, G. Gutierrez, M. Zhao, A. Escobedo, G. Rossini, S.E. Harris, W. Gallwitz, K.B. Kim, S. Hu, C.M. Crews, G.R. Mundy
Catecholamines and α1-adrenergic receptors (α1-ARs) cause cardiac hypertrophy in cultured myocytes and transgenic mice, but heart size is normal in single KOs of the main α1-AR subtypes, α1A/C and α1B. Here we tested whether α1-ARs are required for developmental cardiac hypertrophy by generating α1A/C and α1B double KO (ABKO) mice, which had no cardiac α1-AR binding. In male ABKO mice, heart growth after weaning was 40% less than in WT, and the smaller heart was due to smaller myocytes. Body and other organ weights were unchanged, indicating a specific effect on the heart. Blood pressure in ABKO mice was the same as in WT, showing that the smaller heart was not due to decreased load. Contractile function was normal by echocardiography in awake mice, but the smaller heart and a slower heart rate reduced cardiac output. α1-AR stimulation did not activate extracellular signal–regulated kinase (Erk) and downstream kinases in ABKO myocytes, and basal Erk activity was lower in the intact ABKO heart. In female ABKO mice, heart size was normal, even after ovariectomy. Male ABKO mice had reduced exercise capacity and increased mortality with pressure overload. Thus, α1-ARs in male mice are required for the physiological hypertrophy of normal postnatal cardiac development and for an adaptive response to cardiac stress.
Timothy D. O’Connell, Shinji Ishizaka, Akihiro Nakamura, Philip M. Swigart, M.C. Rodrigo, Gregory L. Simpson, Susanna Cotecchia, D. Gregg Rokosh, William Grossman, Elyse Foster, Paul C. Simpson