Clostridium botulinum C3bot mediated effects on cytokine-induced psoriasis-like phenotype in full-thickness skin model.

Clostridium botulinum C3 exoenzyme (C3bot) exclusively inhibits RhoA, B and C by ADP-ribosylation and is therefore used as a cell-permeable tool for investigating the cellular role of these Rho-GTPases. Rho-GTPases represent a molecular switch integrating different receptor signalling to downstream cascades including transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton and cell proliferation. C3bot-induced inhibition of RhoA leads to reorganization of the actin cytoskeleton, morphological changes, and inhibition of cell proliferation as well as modulation of inflammatory response. In this study, we characterized the C3bot-mediated effects on a full-thickness skin model exhibiting a psoriasis-like phenotype through the addition of cytokines. Indeed, after the addition of cytokines, a decrease in epidermal thickness, parakeratosis, and induction of IL-6 was detected. In the next step, it was studied whether C3bot caused a reduction in the cytokine-induced psoriasis-like phenotypes. Basal addition of C3bot after cytokine induction of the full-thickness skin models caused less epidermal thinning and reduced IL-6 abundance. Simultaneous basal incubation with cytokines and C3bot, IL-6 abundance was inhibited, but epidermal thickness was only moderately affected. When C3bot was added apically to the skin model, IL-6 abundance was reduced, but no further effects on the psoriasis-like phenotype of the epidermis were observed. In summary, C3bot inhibits the cytokine-induced expression of IL-6 and thus may have an impact on the pro-inflammatory immune response in the psoriasis-like phenotype.

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to ß1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin.

Anti-proliferative Effect of C3 Exoenzyme in Fibroblasts is Mediated by c-Jun Phosphorylation.

The ADP-ribosyltransferase C3 exoenzyme from C. botulinum selectively inactivates Rho and is therefore often used as an inhibitor for investigations on Rho signaling. Previous studies of our group revealed that C3 inhibited cell proliferation in HT22 cells accompanied by increased transcriptional activities of Sp1 and c-Jun and reduced levels of cyclin D1, p21 and phosphorylated p38. By use of a p38α-deficient and a p38α-expressing control cell line, the impact of p38 on C3-mediated inhibition of cell proliferation and alterations on MAPK signaling was studied by growth kinetic experiments and Western blot analyses. The cell growth of p38α-expressing cells was impaired by C3, while the p38α-deficient cells did not exhibit any C3-induced effect. The activity of the MKK3/6-p38 MAPK signaling cascade as well as the phosphorylation of c-Jun and JNK was reduced by C3 exclusively in the presence of p38α. Moreover, the activity of upstream MAPKKK TAK1 was lowered in the p38α-expressing cells. These results indicated a resistance of p38α-deficient cells to C3-mediated inhibition of cell growth. This anti-proliferative effect was highly associated with the decreased activity of c-Jun and upstream p38 and JNK MAPK signaling as a consequence of the absence of p38α in these cells.

C3 exoenzyme impairs cell proliferation and apoptosis by altering the activity of transcription factors.

C3 exoenzyme from C. botulinum is an ADP-ribosyltransferase that inactivates selectively RhoA, B, and C by coupling an ADP-ribose moiety. Rho-GTPases are involved in various cellular processes, such as regulation of actin cytoskeleton, cell proliferation, and apoptosis. Previous studies of our group with the murine hippocampal cell line HT22 revealed a C3-mediated inhibition of cell proliferation after 48 h and a prevention of serum-starved cells from apoptosis. For both effects, alterations of various signaling pathways are already known, including also changes on the transcriptional level. Investigations on the transcriptional activity in HT22 cells treated with C3 for 48 h identified five out of 48 transcription factors namely Sp1, ATF2, E2F-1, CBF, and Stat6 with a significantly regulated activity. For validation of identified transcription factors, studies on the protein level of certain target genes were performed. Western blot analyses exhibited an enhanced abundance of Sp1 target genes p21 and COX-2 as well as an increase in phosphorylation of c-Jun. In contrast, the level of p53 and apoptosis-inducing GADD153, a target gene of ATF2, was decreased. Our results reveal that C3 regulates the transcriptional activity of Sp1 and ATF2 resulting downstream in an altered protein abundance of various target genes. As the affected proteins are involved in the regulation of cell proliferation and apoptosis, thus the C3-mediated anti-proliferative and anti-apoptotic effects are consequences of the Rho-dependent alterations of the activity of certain transcriptional factors.

Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.

C3-induced release of neurotrophic factors from Schwann cells - potential mechanism behind its regeneration promoting activity.

Previous studies revealed a peripheral nerve regeneration (PNR)(1) promoting activity of Clostridium botulinum C3(2) exoenzyme or a 26(mer) C-terminal peptide fragment covering amino acids 156-181 (C3(156-181)),(3) when delivered as one-time injection at the lesion site. The current study was performed to 1) investigate if prolonged availability of C3 and C3(156-181) at the lesion site can further enhance PNR in vivo and to 2) elucidate effects of C3 and C3(156-181) on Schwann cells (SCs)(4)in vitro. For in vivo studies, 10 mm adult rat sciatic nerve gaps were reconstructed with the epineurial pouch technique or autologous nerve grafts. Epineurial pouches were filled with a hydrogel containing i) vehicle, ii) 40 µM C3 or iii) 40 µM C3(156-181). Sensory and motor functional recovery was monitored over 12 weeks and the outcome of PNR further analyzed by nerve morphometry. In vitro, we compared gene expression profiles (microarray analysis) and neurotrophic factor expression (western blot analysis) of untreated rat neonatal SCs with those treated with C3 or C3(156-181) for 72 h. Effects on neurotrophic factor expression levels were proven in adult human SCs. Unexpectedly, prolonged delivery of C3 and C3(156-181) at the lesion site did not increase the outcome of PNR. Regarding the potential mechanism underlying their previously detected PNR promoting action, however, 6 genes were found to be commonly altered in SCs upon treatment with C3 or C3(156-181). We demonstrate significant down-regulation of genes involved in glutamate uptake (Eaac1,(5)Grin2a(6)) and changes in neurotrophic factor expression (increase of FGF-2(7) and decrease of NGF(8)). Our microarray-based expression profiling revealed novel C3-regulated genes in SCs possibly involved in the axonotrophic (regeneration promoting) effects of C3 and C3(156-181). Detection of altered neurotrophic factor expression by C3 or C3(156-181) treated primary neonatal rat SCs and primary adult human SCs supports this hypothesis.

Uptake of clostridium botulinum C3 exoenzyme into intact HT22 and J774A.1 cells.

The Clostridium botulinum C3 exoenzyme selectively ADP-ribosylates low molecular weight GTP-binding proteins RhoA, B and C. This covalent modification inhibits Rho signaling activity, resulting in distinct actin cytoskeleton changes. Although C3 exoenzyme has no binding, the translocation domain assures that C3 enters cells and acts intracellularly. C3 uptake is thought to occur due to the high concentration of the C3 enzyme. However, recent work indicates that C3 is selectively endocytosed, suggesting a specific endocytotic pathway, which is not yet understood. In this study, we show that the C3 exoenzyme binds to cell surfaces and is internalized in a time-dependent manner. We show that the intermediate filament, vimentin, is involved in C3 uptake, as indicated by the inhibition of C3 internalization by acrylamide, a known vimentin disruption agent. Inhibition of C3 internalization was not observed by chemical inhibitors, like bafilomycin A, methyl-ß-cyclodextrin, nocodazole or latrunculin B. Furthermore, the internalization of C3 exoenzyme was markedly inhibited in dynasore-treated HT22 cells. Our results indicate that C3 internalization depends on vimentin and does not depend strictly on both clathrin and caveolae.

Vimentin mediates uptake of C3 exoenzyme.

Clostridium botulinum C3 exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 exoenzyme.

Binding of Clostridium botulinum C3 exoenzyme to intact cells.

C3 from Clostridium botulinum (C3) specifically modifies Rho GTPases RhoA, RhoB, and RhoC by mono-ADP-ribosylation. The confined substrate profile of C3 is the basis for its use as pharmacological tool in cell biology to study cellular functions of Rho GTPases. Although C3 exoenzyme does not possess a cell-binding/-translocation domain, C3 is taken up by intact cells via an unknown mechanism. In the present work, binding of C3 to the hippocampus-derived HT22 cells and J774A.1 macrophages was characterized. C3 bound concentration-dependent to HT22 and J774A.1 cells. Pronase treatment of intact cells significantly reduced both C3 binding and C3 cell entry. Removal of sugar residues by glycosidase F treatment resulted in an increased binding of C3, but a reduced cell entry. To explore the involvement of phosphorylation in the binding process of C3, intact HT22 and J774A.1 cells were pre-treated with vanadate prior to incubation with C3. Inhibition of de-phosphorylation by vanadate resulted in an increased binding of C3. To differentiate between intracellular and extracellular phosphorylation, intact cells were treated with CIP (calf intestine phosphatase) to remove extracellular phosphate residues. The removal of phosphate residues resulted in a strong reduction in binding of C3 to cells. In sum, the C3 membranous binding partner is proteinaceous, and the glycosylation as well as the phosphorylation state is critical for efficient binding of C3.

Inhibition of macrophage migration by C. botulinum exoenzyme C3.

C3-like exoenzymes are produced by various microorganism including Clostridium botulinum (C3bot), Bacillus cereus and Staphylococcus aureus. C3bot is the prototype of C3-like exoenzymes that specifically ADP-ribosylates and thereby inactivates Rho(A/B/C). C3-like exoenzymes are not yet regarded as virulence factors, as the lack of cell entry domains results in a poor accessibility of the C3-like exoenzymes to cells. In this study, the sensitivity of various cell lines to C3bot has been reinvestigated. Primary monocytes as well as cultured macrophage-like cells including J774A.1 cells and RAW macrophages exhibit a tenfold higher sensitivity to C3bot than fibroblasts and epithelial cells. RhoA ADP-ribosylation by C3bot resulted in the formation of pronounced bipolar protrusions based on defective tail retraction. The formation of bipolar protrusion resulted in inhibited macrophage migration. These findings suggested that macrophages appear to be target cells of C3bot. Migration of macrophage is a prerequiste for their recruitment to the site of pathogen invasion or tissue damage. Inhibition of macrophage migration likely preserves the survival of C3-producing microorganisms. The observations of this study reinforce the paradigm of a role of C3-like exoenzymes as virulence factors.

Distinct biological activities of C3 and ADP-ribosyltransferase-deficient C3-E174Q.

Low-molecular-weight GTP-binding proteins of the Rho family control the organization of the actin cytoskeleton in eukaryotic cells. Dramatic reorganization of the actin cytoskeleton is caused by the C3 exoenzyme derived from Clostridium botulinum (C3), based on ADP-ribosylation of RhoA/B/C. In addition, wild-type as well as ADP-ribosyltransferase-deficient C3-E174Q induce axonal outgrowth of primary murine hippocampal neurons and prevent growth cone collapse, indicating a non-enzymatic mode of action. In this study, we compared the effects of C3-E174Q and wild-type C3 in the murine hippocampal cell line HT22. Treatment of HT22 cells with C3 resulted in Rho ADP-ribosylation and cell rounding. The ADP-ribosyltransferase-deficient mutant C3-E174Q did not induce either Rho ADP-ribosylation or morphological changes. C3 as well as C3-E174Q treatment resulted in growth arrest, reduced expression of cyclin D levels, and increased expression of RhoB, a negative regulator of cell-cycle progression. Serum starvation induced apoptosis in HT22 cells, as determined on the basis of increased expression of caspase-9 and Bax. C3 but not C3-E174Q protected serum-starved HT22 cells from apoptosis. This is the first study separating ADP-ribosyltransferase-dependent from ADP-ribosyltransferase-independent effects of C3. While morphological changes and anti-apoptotic activity strictly depend on ADP-ribosyltransferase activity, the anti-proliferative effects are independent of ADP-ribosyltransferase activity.

Evidence for involvement of the vitamin D receptor gene in idiopathic short stature via a genome-wide linkage study and subsequent association studies.

Stature is a highly heritable trait under both polygenic and major gene control. We aimed to identify genetic regions linked to idiopathic short stature (ISS) in childhood, through a whole genome scan in 92 families each with two affected children with ISS, including constitutional delay of growth and puberty and familial short stature. Linkage analysis was performed for ISS, height and bone age retardation. Chromosome 12q11 showed significant evidence of linkage to ISS and height (maximum non-parametric multipoint LOD scores 3.18 and 2.31 at 55-58 cM, between D12S1301 and D12S1048), especially in sister-sister pairs (LOD score of 1.9 for ISS in 22 pairs). These traits were also linked to chromosomes 1q12 and 2q36. The region on chromosome 12q11 had previously shown significant linkage to adult stature in several genome scans and harbors the vitamin D receptor gene, which has been associated with variation in height. A single nucleotide polymorphism (SNP) (rs10735810, FokI), which leads to a functionally relevant alteration at the protein level, showed preferential transmission of the transcriptionally more active G-allele to affected children (P=0.04) and seems to be responsible for the observed linkage (P=0.05, GIST test). Bone age retardation showed moderate linkage to chromosomes 19p11-q11 and 7p14 (LOD scores 1.69 at 57 cM and 1.42 at 50 cM), but there was no clear overlap with linkage regions for stature. In conclusion, we identified significant linkage, which might be due to a functional SNP in the vitamin D receptor (VDR) gene and could be responsible for up to 34% of ISS cases in the population.

Effects of single non-ortho, mono-ortho, and di-ortho chlorinated biphenyls on human sperm functions in vitro.

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants in the environment. They are present in body fluids associated with reproduction such as follicular fluid, seminal fluid and cervical mucus. Most PCB effects are mediated through the aryl hydrocarbon receptor, which is present in human spermatozoa. Additionally, PCBs may alter various biochemical reactions, such as calcium homeostasis. Therefore we investigated the effects of single non-ortho PCB 126, mono-ortho PCB 118, and di-ortho PCB 153 on human sperm motility, vitality, and calcium-dependent acrosome reaction (AR) in vitro. Human spermatozoa were either treated with different single PCB congeners or their combinations for 5 h at 37 degrees C (spontaneous AR), or for 16 h at room temperature and 4 degrees C (induced AR). Motility was measured after 5 h of incubation. Compared with the controls, PCB exposure had no effects on the percentage of living acrosome reacted spermatozoa, vitality, and motility. There was no difference in the inducibility of the AR between treatment groups and the respective controls after long term incubation. The PCB concentrations used were far higher than those found in cervical mucus or seminal fluid. In vivo effects of PCB congeners on human ejaculated spermatozoa seem to be unlikely. However these results cannot be easily transferred to the in vivo situation, because individual susceptibility has to be considered, and there is no information about synergistic or additive effects with other chemicals present in the male and female reproductive tract.

Children with idiopathic short stature are poor eaters and have decreased body mass index.

OBJECTIVE: In children with idiopathic short stature (ISS), studies investigating body mass index (BMI) or parameters of satiety regulation are scarce, and studies analyzing eating behavior are lacking. METHODS: We recruited 214 children (123 index cases and 91 siblings) with ISS from 123 families. Affected children had to have a body height <5th percentile, or, in the case of siblings, the body height of 1 child had to be <5th percentile and the other <15th percentile. Medical histories were recorded by using structured and standardized interviews. Eating behavior was assessed by using the Child Eating Behavior Questionnaire. Percent energy intake as fat was assessed by using the Leeds Food Frequency Questionnaire. Endocrine markers of body weight regulation (leptin, ghrelin) were determined in serum. RESULTS: Compared with population norms, BMI was significantly lower (mean: -0.33 standard deviation score). Furthermore, there was decreased food responsiveness (mean Child Eating Behavior Questionnaire score: 1.9; population mean: 2.4), reduced enjoyment of food (3.2 vs 3.9), emotional undereating (2.6 vs 3.0), lower desire to drink (2.0 vs 2.8), and increased fussiness over food (3.2 vs 2.9). When the sample was subdivided into the 2 groups of "good" and "poor" eaters according to the mothers' assessment of the current eating behavior, reduction in BMI as well as the behavioral characteristics already delineated in the total sample were found to be even more consistent in the subgroup of poor eaters. In the total sample of our children, as well as in both subgroups, serum leptin (adjusted for gender, BMI, and Tanner stage) was found to be moderately raised but did not differ between poor and good eaters. Total serum ghrelin was not different between poor and good eaters. CONCLUSIONS: Our clinical, behavioral, and endocrinologic findings in patients with ISS point to an altered eating behavior that possibly contributes to their short stature.

Ghrelin receptor gene: identification of several sequence variants in extremely obese children and adolescents, healthy normal-weight and underweight students, and children with short normal stature.

GH secretagogue receptor (GHSR, ghrelin receptor) is involved in regulation of body weight and GH secretion. We initially analyzed two single-nucleotide polymorphisms of the GHSR in up to 184 extremely obese children and adolescents and up to 184 healthy underweight students. The frequency of the 171T allele of rs495225 was higher in our obese samples (75.0%) than in the underweight individuals (70.2%; nominal P = 0.14). This trend could not be substantiated in an additional association study in 270 obese and 145 underweight and normal weight individuals and in a transmission disequilibrium test based on 387 obesity trios (transmission rate of 171T, 51.8%; nominal P = 0.53). Additionally, the coding region of GHSR was systematically screened, and seven sequence variants were identified in 93 obese, 96 normal weight, and 94 underweight individuals and 43 children with short normal stature (SNS). Five silent single-nucleotide polymorphisms showed similar genotype frequencies in the different weight groups and SNS children (all nominal P > 0.3). Two novel missense variants were detected only in one obese carrier and one SNS child, respectively. In conclusion, we did not obtain conclusive evidence for an involvement of the ghrelin receptor gene in body weight regulation or SNS in our study groups.