Biliverdin modulates the expression of C5aR in response to endotoxin in part via mTOR signaling

Macrophages play a crucial role in the maintenance and resolution of inflammation and express a number of pro- and anti-inflammatory molecules in response to stressors. Among them, the complement receptor 5a (C5aR) plays an integral role in the development of inflammatory disorders. Biliverdin and bilirubin, products of heme catabolism, exert anti-inflammatory effects and inhibit complement activation. Here, we define the effects of biliverdin on C5aR expression in macrophages and the roles of Akt and mammalian target of rapamycin (mTOR) in these responses. Biliverdin administration inhibited lipopolysaccharide (LPS)-induced C5aR expression (without altering basal expression), an effect partially blocked by rapamycin, an inhibitor of mTOR signaling. Biliverdin also reduced LPS-dependent expression of the pro-inflammatory cytokines TNF-alpha and IL-6. Collectively, these data indicate that biliverdin regulates LPS-mediated expression of C5aR via the mTOR pathway, revealing an additional mechanism underlying biliverdin's anti-inflammatory effects.

Laser capture microdissection and multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in human kidney disease

Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate “real time” gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-β in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.

Growth defect in Grg5 null mice is associated with reduced Ihh signaling in growth plates

Gene-targeted disruption of Grg5, a mouse homologue of Drosophila groucho (gro), results in postnatal growth retardation in mice. The growth defect, most striking in approximately half of the Grg5 null mice, occurs during the first 4-5 weeks of age, but most mice recover retarded growth later. We used the nonlinear mixed-effects model to fit the growth data of wild-type, heterozygous, and Grg5 null mice. On the basis of preliminary evidence suggesting an interaction between Grg5 and the transcription factor Cbfa1/Runx2, critical for skeletal development, we further investigated the skeleton in the mice. A long bone growth plate defect was identified, which included shorter zones of proliferative and hypertrophic chondrocytes and decreased trabecular bone formation. This decreased trabecular bone formation is likely caused by a reduced recruitment of osteoblasts into the growth plate region of Grg5 null mice. Like the growth defect, the growth plate and trabecular bone abnormality improved as the mice grew older. The growth plate defect was associated with reduced Indian hedgehog expression and signaling. We suggest that Grg5, a transcriptional coregulator, modulates the activities of transcription factors, such as Cbfa1/Runx2 in vivo to affect Ihh expression and the function of long bone growth plates.

M. bovis BCG induced expression of COX-2 involves nitric oxide-dependent and -independent signaling pathways

In a multifaceted immunity to mycobacterial infection, induced expression of cyclooxygenase-2 (COX-2) by Mycobacterium bovis bacillus Calmette-Guerin (BCG) may act as an important influencing factor for the effective host immunity. We here demonstrate that M. bovis BCG-triggered TLR2-dependent signaling leads to COX-2 and PGE2 expression in vitro in macrophages and in vivo in mice. Further, the presence of PGE2 could be demonstrated in sera or cerebrospinal fluid of tuberculosis patients. The induced COX-2 expression in macrophages is dependent on NF-kappa B activation, which is mediated by inducible NO synthase (iNOS)/NO-dependent participation of the members of Notch1-PI-3K signaling cascades as well as iNOS-independent activation of ERK1/2 and p38 MAPKs. Inhibition of iNOS activity abrogated the M. bovis BCG ability to trigger the generation of Notch1 intracellular domain (NICD), a marker for Notch1 signaling activation, as well as activation of the PI-3K signaling cascade. On the contrary, treatment of macrophages with 3-morpholinosydnonimine, a NO donor, resulted in a rapid increase in generation of NICD, activation of PI-3K pathway, as well as the expression of COX-2. Stable expression of NICD in RAW 264.7 macrophages resulted in augmented expression of COX-2. Further, signaling perturbations suggested the involvement of the cross-talk of Notch1 with members with the PI-3K signaling cascade. These results implicate the dichotomous nature of TLR2 signaling during M. bovis BCG-triggered expression of COX-2. In this perspective, we propose the involvement of iNOS/NO as one of the obligatory, early, proximal signaling events during M. bovis BCG-induced COX-2 expression in macrophages.

Uropathogenic Escherichia Coli engages CD14-dependent signaling to enable bladder-macrophage-dependent control of acute urinary tract infection

Background CD14, a coreceptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to gram-negative bacteria. Despite the central role of CD14 in the inflammatory response to lipopolysaccharide and other microbial products and in the dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown. Methods We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14−/− mice and RNA sequencing to define the CD14-dependent transcriptional signature and the role of CD14 in host defense against UTI in the bladder. Results UPEC induced the upregulation of Cd14 and the monocyte/macrophage-related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT mice, compared with Cd14−/− mice. Exacerbation of infection in Cd14−/− mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and interleukin 17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens. Conclusions This study identifies new host protective and signaling roles for CD14 in the bladder during UPEC UTI.

Kidney Induction: control by Notch, Wnt and GDNF/Ret signalling

The permanent mammalian kidney (metanephros) develops as a result of complex reciprocal tissue interactions between a ureteric epithelium and the renal mesenchyme. The overall goal of the research in this thesis was to gain data that will eventually help in elucidating the formation of congenital renal malformations. The experiments in my thesis aimed to reveal the mechanisms by which Notch, Wnt and GDNF/Ret signalling pathways regulate the development of functional kidney. The function of Notch pathway was studied by a transgenic mouse model, where it was shown that overactivation of Notch signalling disturbs kidney development and alters the expression of Gdnf and Ret/GFRa1. This indicates that Notch signalling interplays with GDNF/Ret in the regulation of the primary ureteric budding and its subsequent branching. The data also suggested that strict spatio-temporal regulation of these two pathways is required for determination of ureteric tip-identity, which appeared to be crucial for the branch formation. The function of Wnt signalling in the ureteric morphogenesis was studied by in vivo and in vitro methods to show that a canonical pathway is required for ureteric branching. Stabilisation and deletion of the canonical pathway mediator, b-catenin specifically in the ureteric epithelium result in renal aplasia/hypodysplasia. These defects originate from severe blockage of ureteric branching due to the disrupted Ret signalling. Consequently, ureteric tip specific markers are lost and ureteric stalk identity is expanded throughout the whole epithelium. Thus, the data demonstrates that the Wnt/b-catenin pathway plays an essential role in the patterning and branching of the ureteric epithelium. A novel in vitro method was generated and utilised in nephron induction studies to reveal the mechanisms through which nephrogenesis is induced. Transient GSK3 inhibition results in stabilisation of b-catenin in the isolated renal mesenchyme, which efficiently triggers nephron formation. Also genetic stabilisation of b-catenin specifically in the mesenchyme results in spontaneous nephrogenesis. The results show that activation of the canonical Wnt pathway is sufficient to initiate nephrogenesis, and suggest that this pathway mediates the nephron induction in murine kidney mesenchymes. Taken together, this thesis demonstrates Notch and Wnt signalling pathways as novel regulators of ureteric branching morphogenesis, and that activation of the canonical Wnt pathway is sufficient for nephron induction. The studies also indicate that the Notch and Wnt pathways cross-talk with GDNF/Ret signalling in the patterning of ureteric epithelium.

Defining the role of common variation in the genomic and biological architecture of adult human height

Using genome-wide data from 253,288 individuals, we identified 697 variants at genome-wide significance that together explained one-fifth of the heritability for adult height. By testing different numbers of variants in independent studies, we show that the most strongly associated approximately 2,000, approximately 3,700 and approximately 9,500 SNPs explained approximately 21%, approximately 24% and approximately 29% of phenotypic variance. Furthermore, all common variants together captured 60% of heritability. The 697 variants clustered in 423 loci were enriched for genes, pathways and tissue types known to be involved in growth and together implicated genes and pathways not highlighted in earlier efforts, such as signaling by fibroblast growth factors, WNT/beta-catenin and chondroitin sulfate-related genes. We identified several genes and pathways not previously connected with human skeletal growth, including mTOR, osteoglycin and binding of hyaluronic acid. Our results indicate a genetic architecture for human height that is characterized by a very large but finite number (thousands) of causal variants.

Identification of the Meckel syndrome gene (MKS1) exposes a novel ciliopathy

Meckel syndrome (MKS, MIM 249000) is an autosomal recessive developmental disorder causing death in utero or shortly after birth. The hallmarks of the disease are cystic kidney dysplasia and fibrotic changes of the liver, occipital encephalocele with or without hydrocephalus and polydactyly. Other anomalies frequently seen in the patients are incomplete development of the male genitalia, club feet and cleft lip or palate. The clinical picture has been well characterized in the literature while the molecular pathology underlying the disease has remained unclear until now. In this study we identified the first MKS gene by utilizing the disease haplotypes in Finnish MKS families linked to the MKS1 locus on chromosome 17q23 (MKS1) locus. Subsequently, the genetic heterogeneity of MKS was established in the Finnish families. Mutations in at least four different genes can cause MKS. These genes have been mapped to the chromosomes 17q23 (MKS1), 11q13 (MKS2), 8q22 (MKS3) and 9q33 (MKS4). Two of these genes have been identified so far: The MKS1 gene (this work) and the MKS3 gene. The identified MKS1 gene was initially a novel human gene which is conserved among species. We found three different MKS mutations, one of them being the Finnish founder mutation. The information available from MKS1 orthologs in other species convinced us that the MKS1 gene is required for normal ciliogenesis. Defects of the cilial system in other human diseases and model organisms actually cause phenotypic features similar to those seen in MKS patients. The MKS3 (TMEM67) gene encodes a transmembrane protein and the gene maps to the syntenic Wpk locus in the rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. The available information from these two genes suggest that MKS1 would encode a structural component of the centriole required for normal ciliary functions, and MKS3 would be a transmembrane component most likely required for normal ciliary sensory signaling. The MKS4 locus was localized to chromosme 9q32-33 in this study by using an inbred Finnish family with two affected and two healthy children. This fourth locus contains TRIM32 gene, which is associated to another well characterized human ciliopathy, Bardet Biedl syndrome (BBS). Future studies should identify the MKS4 gene on chromosome 9q and confirm if there are more than two genes causing MKS Finnish families. The research on critical signaling pathways in organogenesis have shown that both Wnt and Hedgehog pathways are dependent on functional cilia. The MKS gene products will serve as excellent model molecules for more detailed studies of the functional role of cilia in organogenesis in more detail.

Mutation analysis of TGF-β signaling pathway genes among Finnish patients with primary pulmonary hypertension and hereditary hemorrhagic telangiectasia

Primary pulmonary hypertension (PPH), or according to the recent classification idiopathic pulmonary hypertension (IPAH), is a rare, progressive disease of pulmonary vasculature leading to pulmonary hypertension and right heart failure. Most of the patients are sporadic but in about 6% of cases the disease is familial (FPPH). In 2000 two different groups identified the gene predisposing to PPH. This gene, Bone morphogenetic protein receptor type 2 (BMPR2), encodes a subunit of transforming growth factor β (TGF-β) receptor complex. There is a genetic connection between PPH and hereditary hemorrhagic telangiectasia (HHT), a bleeding disorder characterized by local telangiectasias and sometimes with pulmonary hypertension. In HHT, mutations in ALK1 (activin like kinase type 1) and Endoglin, another members of the TGF-β signaling pathway are found. In this study we identified all of the Finnish PPH patients for the years 1986-1999 using the hospital discharge registries of Finnish university hospitals. During this period we found a total of 59 confirmed PPH patients: 55 sporadic and 4 familial representing 3 different families. In 1999 the prevalence of PPH was 5.8 per million and the annual incidence varied between 0.2-1.3 per million. Among 28 PPH patients studied, heterozygous BMPR2 mutations were found in 12% (3/26) of sporadic patients and in 33% of the PPH families (1/3). All the mutations found were different. Large deletions of BMPR2 were excluded by single-stranded chain polymomorphism analysis. As a candidate gene approach we also studied ALK1, Endoglin, Bone Morphogenetic Receptor Type IA (BMPR1A or ALK3), Mothers Against Decapentaplegic Homolog 4 (SMAD4) and Serotonine Transporter Gene (SLC6A4) using single-strand conformational polymorphism (SSCP) analysis and direct sequencing. Among patients and family members studied, we found two mutations in ALK1 in two unrelated samples. We also identified all the HHT patients treated at the Department of Otorhinolaryngology at Helsinki University Central Hospital between the years of 1990-2005 and 8 of the patients were studied for Endoglin and ALK1 mutations using direct sequencing. A total of seven mutations were found and all the mutations were different. The absence of a founder mutation in the Finnish population in both PPH and HHT was somewhat surprising. This suggests that the mutations of BMPR2, ALK1 and Endoglin are quite young and the older mutations have been lost due to repetitive genetic bottlenecks and/or negative selection. Also, other genes than BMPR2 may be involved in the pathogenesis of PPH. No founder mutations were found in PPH or HHT and thus no simple genetic test is available for diagnostics.

Pathogen-Induced Defense Signaling and Signal Crosstalk in Arabidopsis

Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.

VEGFR-2 and VEGFR-3 Specific Signaling in Lymphangiogenesis and Angiogenesis

The circulatory system consists of the blood and lymphatic vessels. While blood vessels transport oxygen, cells, and nutrients to tissues, the lymphatic vessels collect fluid, cells, and plasma proteins from tissues to return back to the blood circulation. Angiogenesis, the growth of new blood vessels from pre-existing ones, is an important process involved in several physiological conditions such as inflammation, wound healing, and embryonic development. Furthermore, angiogenesis is found in many pathological conditions such as atherosclerosis and the growth and differentiation of solid tumors. Many tumor types spread via lymphatic vessels to form lymph node metastasis. The elucidation of the molecular players coordinating development of the vascular system has provided an array of tools for further insight of the circulatory system. The discovery of the Vascular Endothelial Growth Factor (VEGF) family members and their tyrosine kinase receptors (VEGFRs) has facilitated the understanding of the vasculature in different physiological and pathological situations. The VEGFRs are expressed on endothelial cells and mediate the growth and maintenance of both the blood and lymphatic vasculatures. This study was undertaken to address the role of VEGFR-2 specific signaling in maturation of blood vessels during neoangiogenesis and in lymphangiogenesis. We also wanted to differentiate between VEGFR-2 and VEGFR-3 specific signaling in lymphangiogenesis. We found that specific VEGFR-2 stimulation alone by gene therapeutic methods is not sufficient for production of mature blood vessels. However, VEGFR-2 stimulation in combination with expression of platelet-derived growth factor D (PDGF-D), a recently identified member of the PDGF growth factor family, was capable of stabilizing these newly formed vessels. Signaling through VEGFR-3 is crucial during developmental lymphangiogenesis, but we showed that the lymphatic vasculature becomes independent of VEGFR-3 signaling after the postnatal period. We also found that VEGFR-2 specific stimulation cannot rescue the loss of lymphatic vessels when VEGFR-3 signaling is blocked and that VEGFR-2 specific signals promote lymphatic vessel enlargement, but are not involved in vessel sprouting to generate new lymphatic vessels in vivo, in contrast to the VEGFR-2 dependent sprouting observed in blood vessels. In addition, we compared the inhibitory effects of a small molecular tyrosine kinase inhibitor of VEGFR-2 vs. VEGFR-3 specific signaling in vitro and in vivo. Our results showed that the tyrosine kinase inhibitor could equally affect physiological and pathological processes dependent on VEGFR-2 and VEGFR-3 driven angiogenesis or lymphangiogenesis. These results provide new insights into the VEGFR specific pathways required for pre- and postnatal angiogenesis as well as lymphangiogenesis, which could provide important targets and therapies for treatment of diseases characterized by abnormal angiogenesis or lymphangiogenesis.

SOCS3 expression induced by PIM2 requires PKC and PI3K signaling

Initiation of proinflammatory host immunity in response to infection represents as a key event in effective control and containment of the pathogen at the site of infection as well as in elicitation of robust immune memory responses. In the current investigation, we demonstrate that an integral cell wall antigen of the mycobacterial envelope, Phosphatidyl-myo-inositol dimannosides (PIM2) triggers Suppressor of cytokine signaling (SOCS) 3 expression in macrophages in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Data derived from signaling perturbations suggest the involvement of phosphoinositide-3 kinase (PI3K) and protein kinase C (PKC) signaling pathways during PIM2 induced SOCS3 expression. Further, pharmacological inhibition of ERK1/2, but not of p38 MAP kinase or JNK abrogated the induced expression of SOCS3. The PIM2 induced activation of ERK1/2 was dependent on the activation of PI3K or PKC signaling which in turn regulated p65 nuclear factor -kappa B (NF-kappa B) nuclear translocation. Overall, current study delineates the role for PI3K-PKC axis and ERK1/2 signaling as key signaling events during PIM2 induced SOCS3 expression in macrophages.