Suppressors of cytokine signalling (SOCS): putative modulators of cytokine bioactivity in health and disease

Cytokines are important modulators of homeostatic processes such as development, haematopoiesis and host defence, A recently identified family of proteins, the supressors of cytokine signalling (SOCS) act as negative regulators of the key cytokine-activated signalling pathway, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) cascade, In the current review, the discovery, structural features, regulation of expression, mechanisms of JAK/STAT inhibition and putative role in health and disease of the SOCS family are discussed.

Smooth Muscle Cells Differentiated from Reprogrammed Embryonic Lung Fibroblasts Through DKK3 Signalling are Potent for Tissue Engineering of Vascular Grafts

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited.

Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state.

Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft.

Conclusions: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

Host cell kinases, α5 and β1 integrins, and Rac1 signalling on the microtubule cytoskeleton are important for non-typable Haemophilus influenzae invasion of respiratory epithelial cells

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase C?1 inhibition but was not affected by protein kinase inhibition. We also found that a5 and ß1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.

Advanced glycation endproduct modified basement membrane attenuates endothelin-1 induced [Ca2+]i signalling and contraction in retinal microvascular pericytes

PURPOSE: To assess the effects of advanced glycation endproduct (AGE) modification of vascular basement membrane (BM) on endothelin-1 (Et-1) induced intracellular [Ca2+] ([Ca2+]i) homeostasis and contraction in retinal microvascular pericytes (RMP). METHODS: RMPs were isolated from bovine retinal capillaries and propagated on AGE modified BM extract (AGE-BM) or non-modified native BM. Cytosolic Ca2+ was estimated using fura-2 microfluorimetry and cellular contraction determined by measurement of planimetric cell surface area. ETA receptor mRNA and protein expression was assessed by real time RT-PCR and western blotting, respectively. RESULTS: Exogenous endothelin-1 (Et-1) evoked rises in [Ca2+]i and contraction in RMPs were found to be mediated entirely through ETA receptor (ETAR) activation. Both peak and plateau phases of the Et-1 induced [Ca2+]i response and contraction were impaired in RMPs propagated on AGE modified BM. ETAR mRNA expression remained unchanged in RMPs exposed to native or AGE-BM, but protein expression for ETAR (66 kDa) was lower in the AGE exposed cells. CONCLUSIONS: These results suggest that substrate derived AGE crosslinks can influence RMP physiology by mechanisms which include disruption of ETA receptor signalling. AGE modification of vascular BMs may contribute to the retinal hemodynamic abnormalities observed during diabetes.

Role of ion channels and subcellular Ca2+ signalling in arachidonic acid induced dilation of pressurised retinal arterioles

Purpose: To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Methods: Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacological agents inhibiting Ca2+ sparks and oscillations and K+ channels. Subcellular Ca2+ signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Results: AA dilated pressurised retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. AA-induced dilation was associated with an inhibition of subcellular Ca2+ signals. Interventions known to block Ca2+ sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. AA accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous BK currents, but only at positive membrane potentials. Pharmacological inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. AA suppressed L-type Ca2+ currents. Conclusions: These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca2+ signalling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca2+ channel activity. AA actions on K+ currents are inconsistent with a model in which K+ channels contribute to the vasodilator effects of AA.

Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2.

Prenatal hypoxia leads to increased muscle sympathetic nerve activity, sympathetic hyperinnervation, premature blunting of NPY signalling and hypertension in adult life

Adverse conditions prenatally increase the risk of cardiovascular disease, including hypertension. Chronic hypoxia in utero (CHU) causes endothelial dysfunction, but whether sympathetic vasoconstrictor nerve functioning is altered is unknown. We, therefore, compared in male CHU and control (N) rats muscle sympathetic nerve activity, vascular sympathetic innervation density, and mechanisms of sympathetic vasoconstriction. In young (Y)-CHU and Y-N rats (≈3 months), baseline arterial blood pressure was similar. However, tonic muscle sympathetic nerve activity recorded focally from arterial vessels of spinotrapezius muscle had higher mean frequency in Y-CHU than in Y-N rats (0.56±0.075 versus 0.33±0.036 Hz), and the proportions of single units with high instantaneous frequencies (1–5 and 6–10 Hz) being greater in Y-CHU rats. Sympathetic innervation density of tibial arteries was ≈50% greater in Y-CHU than in Y-N rats. Increases in femoral vascular resistance evoked by sympathetic stimulation at low frequency (2 Hz for 2 minutes) and bursts at 20 Hz were substantially smaller in Y-CHU than in Y-N rats. In Y-N only, the neuropeptide Y Y1-receptor antagonist BIBP3226 attenuated these responses. By contrast, baseline arterial blood pressure was higher in middle-aged (M)-CHU than in M-N rats (≈9 months; 139±3 versus 126±3 mmHg, respectively). BIBP3226 had no effect on femoral vascular resistance increases evoked by 2 Hz or 20 Hz bursts in M-N or M-CHU rats. These results indicate that fetal programming induced by prenatal hypoxia causes an increase in centrally generated muscle sympathetic nerve activity in youth and hypertension by middle age. This is associated with blunting of sympathetically evoked vasoconstriction and its neuropeptide Y component that may reflect premature vascular aging and contribute to increased risk of cardiovascular disease

CaV3.1 T-Type Ca2+ Channels Contribute to Myogenic Signalling in Rat Retinal Arterioles

Purpose: Although L-type Ca2+ channels are known to play a key role in the myogenic reactivity of retinal arterial vessels, the involvement of other types of voltage-gated Ca2+ channels in this process remains unknown. In the present study we have investigated the contribution of T-type Ca2+ channels to myogenic signalling in arterioles of the rat retinal microcirculation.

Methods: Confocal immunolabelling of wholemount preparations was used to investigate the localisation of CaV3.1-3 channels in retinal arteriolar smooth muscle cells. T-type currents and the contribution of T-type channels to myogenic signalling were assessed by whole-cell patch-clamp recording and pressure myography of isolated retinal arteriole segments.

Results: Strong immunolabelling for CaV3.1 was observed on the plasma membrane of retinal arteriolar smooth muscle cells. In contrast, no expression of CaV3.2 or CaV3.3 could be detected in retinal arterioles, although these channels were present on glial cell end feet surrounding the vessels and retinal ganglion cells, respectively. TTA-A2 sensitive T-type currents were recorded in retinal arteriolar myocytes with biophysical properties distinct from those of the L-type currents present in these cells. Inhibition of T-type channels using TTA-A2 or ML-218 dilated isolated, myogenically active, retinal arterioles.

Conclusions: CaV3.1 T-type Ca2+ channels are functionally expressed on arteriolar smooth muscle cells of retinal arterioles and play an important role in myogenic signalling in these vessels. The work has important implications concerning our understanding of the mechanisms controlling blood flow autoregulation in the retina and its disruption during ocular disease.

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008

Brain-derived neurotrophic factor and adenosine signalling on amyloid-β peptide induced toxicity : impact on hippocampal function

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

CD40 signalling in platelet function

Le CD40 est un membre de la famille des récepteurs du facteur de nécrose tumorale ("Tumour necrosis factor", TNF), initialement identifié sur des cellules de carcinome de la vessie. L'interaction du CD40 avec son ligand (CD40L) est d'une importance cruciale pour le développement des cellules B et de la commutation d'isotype au cours de la réponse immunitaire acquise. L'expression du complexe CD40/CD40L était initialement cru d'être limiter aux cellules du système immunitaire, mais aujourd'hui il est bien connu que ce complexe est également exprimé sur les cellules du système circulatoire et vasculaire, et est impliqué dans diverses réactions inflammatoires; de sorte que le CD40L est maintenant considéré comme une molécule thrombo-inflammatoire prédictive des événements cardiovasculaires. Les plaquettes expriment constitutivement le CD40, alors que le CD40L n'est exprimé que suite à leur l'activation. Il est ensuite clivé en sa forme soluble (sCD40L) qui représente la majorité du sCD40L en circulation. Il fut démontré que le sCD40L influence l'activation plaquettaire mais son effet exact sur la fonction plaquettaire, ainsi que les mécanismes cellulaires et moléculaires sous-jacents à son action demeurent inconnus. Ainsi, ce projet a été entrepris dans le but d’adresser les objectifs spécifiques suivants: 1) évaluer les effets in vitro du sCD40L sur l'activation et l'agrégation plaquettaire; 2) identifier les récepteurs plaquettaires impliqués dans l’action du sCD40L; 3) élucider les voies signalétiques intracellulaires induits par le sCD40L; 4) évaluer les effets du sCD40L sur la formation de thrombus in vivo. Nous avons trouvé que le sCD40L augmente fortement l'activation et l'agrégation des plaquettes en réponse à de faibles concentrations d'agonistes. Les plaquettes humaines traitées avec une forme mutante du sCD40L qui n'interagit pas avec le CD40, et les plaquettes de souris déficientes en CD40 ne furent pas en mesure d'induire de telles réponses, indiquant que le récepteur principal du sCD40L au niveau des plaquettes est le CD40. En plus, nous avons identifié la présence de plusieurs membres de la famille du facteur associé du récepteur du TNF ("TNF receptor-associated factor", TRAF) dans les plaquettes et nous avons montré que seulement le TRAF2 s'associe avec le CD40 suite à la stimulation par le sCD40L. Nos résultats indiquent aussi que le sCD40L agisse sur les plaquettes au repos par l'entremise de deux voies signalétiques distinctes. La première voie implique l'activation de la petite GTPase Rac1 et de sa cible en aval, soit la protéine kinase p38 activée par le mitogène ("p38 mitogen-activated protein kinase", p38 MAPK ), menant au changement de forme plaquettaire et à la polymérisation de l'actine; alors que la deuxième voie implique l'activation de la cascade signalétique du NF-kB. Par ailleurs, à la suite d'une lésion artérielle induite par le chlorure de fer, le sCD40L exacerbe la formation de thrombus et l'infiltration leucocytaire au sein du thrombus dans les souris du type sauvage, mais pas chez les souris déficientes en CD40. En conclusion, ce projet a permis d'identifier pour la première fois deux voies signalétiques distinctes en aval du CD40 plaquettaire et a permis d'établir leur implication dans l'activation et l'agrégation plaquettaire en réponse au sCD40L. De manière plus importante, ce projet nous a permis d'établir un lien direct entre les niveaux élevés du sCD40L circulant et la formation de thrombus in vivo, tout en soulignant l'importance du CD40 dans ce processus. Par conséquent, l'axe CD40/CD40L joue un rôle important dans l'activation des plaquettes, les prédisposant à une thrombose accrue en réponse à une lésion vasculaire. Ces résultats peuvent expliquer en partie la corrélation entre les taux circulants élevés du sCD40L et l'incidence des maladies cardiovasculaires.

The role of Ken&Barbie in JAK/STAT signalling during development of Drosophila melanogaster

Cell-cell interactions during embryonic development are crucial in the co-ordination of growth, differentiation and maintenance of many different cell types. To achieve this co-ordination each cell must properly translate signals received from neighbouring cells, into spatially and temporally appropriate developmental responses. A surprisingly limited number of signal pathways are responsible for the differentiation of enormous variety of cell types. As a result, pathways are frequently 'reused' during development. Thus, in mammals the JAK/STAT pathway is required during early embryogenesis, mammary gland formation, hematopoiesis and, finally, plays a pivotal role in immune response. In the canonical way, the JAK/STAT pathway is represented by a transmembrane receptor associated with a Janus kinase (JAK), which upon stimulation by an extra-cellular ligand, phosphorylates itself, the receptor and, finally, the signal transducer and activator of transcription (STAT) molecules. Phosphorylated STATs dimerise and translocate to the nucleus where they activate transcription of target genes. The JAK/STAT pathway has been conserved throughout evolution, and all known components are present in the genome of Drosophila melanogaster. Besides hematopoietic and immunity functions, the pathway is also required during development for processes including embryonic segmentation, tracheal morphogenesis, posterior spiracle formation etc. This study describes Drosophila Ken&Barbie (Ken) as a selective regulator of JAK/STAT signalling. ken mutations identified in a screen for modulators of an eye overgrowth phenotype, caused by over-expression of the pathway ligand unpaired, also interact genetically with the pathway receptor domeless (dome) and the transcription factor stat92E. Over-expression of Ken can phenocopy developmental defects known to be caused by the loss of JAK/STAT signalling. These genetic interactions suggest that Ken may function as a negative regulator of the pathway. Ken has C-terminal Zn-finger domain, presumably for DNA binding, and N-terminal BTB/POZ domain, often found in transcriptional repressors. Using EGFP-fused construct expressed in vivo revealed nuclear accumulation of Ken. Therefore, it is proposed that Ken may act as a suppresser of STAT92E target genes. An in vitro assay, termed SELEX, determined that Ken specifically binds to a DNA sequence, with the essential for DNA recognition core overlapping that of STAT92E. This interesting observation suggests that not all STAT92E sites may also allow Ken binding. Strikingly, when effects of ectopic Ken on the expression of putative JAK/STAT pathway target genes were examined, only a subset of the genes tested, namely vvl, trh and kni, were down-regulated by Ken, whereas some others, such as eve and fj, appeared to be unresponsive. Further analysis of vvl, one of the genes susceptible to ectopic Ken, was undertaken. In the developing hindgut, expression of vvl is JAK/STAT pathway dependent, but remains repressed in the posterior spiracles, despite the stimulation of STAT92E by Upd in their primordia. Importantly, ken is also expressed in the developing posterior spiracles. Strikingly, up-regulation of vvl is observed in these tissues in ken mutant embryos. These imply that while ectopic Ken is sufficient to repress the expression of vvl in the hindgut, endogenous Ken is also necessary to prevent its activation in the posterior spiracles. It is therefore conceivable that ectopic vvl expression in the posterior spiracles of the ken mutants may be the result of de-repression of endogenous STAT92E activity. Another consequence of these observations is a fine balance that must exist between STAT92E and Ken activities. Apparently, endogenous level of Ken is sufficient to repress vvl, but not other, as yet unidentified, JAK/STAT pathway targets, whose presumable activation by STAT92E is required for posterior spiracle development as the embryos mutant for dome, the receptor of the pathway, show severe spiracle defects. These defects are also observed in the embryos mis-expressing Ken. Though it is possible that the posterior spiracle phenotype caused by higher levels of Ken results from a JAK/STAT pathway independent activity, it seems to be more likely that Ken acts in a dosage dependent manner, and extra Ken is able to further antagonise JAK/STAT pathway target genes. While STAT92E binding sites required for target gene expression have been poorly characterised, the existence of genome data allows the prediction of candidate STAT92E sites present in target genes promoters to be attempted. When a 6kb region containing the putative regulatory domains flanking the vvl locus are examined, only a single potential STAT92E binding site located 825bp upstream of the translational start can be detected. Strikingly, this site also includes a perfect Ken binding sequence. Such an in silico observation, though consistent with both Ken DNA binding assay in vitro and regulation of STAT92E target genes in vivo, however, requires further analysis. The JAK/STAT pathway is implicated in a variety of processes during embryonic and larval development as well as in imago. In each case, stimulation of the same transcription factor results in different developmental outcomes. While many potential mechanisms have been proposed and demonstrated to explain such pleiotropy, the present study indicates that Ken may represent another mechanism, with which signal transduction pathways are controlled. Ken selectively down-regulates a subset of potential target genes and so modifies the transcriptional profile generated by activated STAT92E - a mechanism, which may be partially responsible for differences in the morphogenetic processes elicited by JAK/STAT signalling during development.

Characterization of the role of Drosophila JAK/STAT signalling in cellular proliferation

Der Janus Kinase / signal transducer and activator of transcription (JAK/STAT) Signal- transduktionsweg wird für viele Entwicklungsvorgänge benötigt und spielt eine zentrale Rolle bei der Hämatopoese und bei der Immunantwort. Obwohl der JAK/STAT-Signalweg in den vergangenen Jahren Gegenstand intensiver Forschung war, erschwert die Redundanz des Signalwegs bei Wirbeltieren genetische Untersuchungen zur Identifizierung derjenigen Mechanismen, die den JAK/STAT-Signalweg regulieren. Der JAK/STAT-Signaltransduktionsweg ist evolutionär konserviert und ebenfalls bei der Taufliege Drosophila melanogaster vorhanden. Im Gegensatz zu Wirbeltieren ist der Signaltransduktionsweg von Drosophila weniger redundant und beinhaltet folgende Hauptkomponenten: den Liganden Unpaired (Upd), den Transmembranrezeptor Domeless (Dome), die einzige JAK-Tyrosinkinase Hopscotch (hop), sowie den Transkriptionsfaktor STAT92E. In der vorliegenden Arbeit wird die Rolle des JAK/STAT-Signalwegs bei der zellulären Proliferation mithilfe der Modellsysteme der Flügel- und der Augen-Imaginalscheiben von Drosophila charakterisiert. "Loss-of-function"- und "Gain-of-function"-Experimente zur Verminderung beziehungs-weise Erhöhung der Signalaktivität zeigten, dass der JAK/STAT-Signalweg eine Rolle bei der zellulären Proliferation der Flügel-Imaginalscheiben spielte, ohne die Zellgröße oder Apoptose zu verändern. Bei der Flügelentwicklung während des zweiten und des frühen dritten Larvalstadiums war die Aktivität des JAK/STAT-Signalwegs sowohl notwendig für die zelluläre Proliferation als auch hinreichend, um Überproliferation anzutreiben. Allerdings änderte sich während der späten dritten Larvalstadien die JAK/STAT-Signalaktivität, sodass endogene STAT92E-Mengen einen anti-proliferativen Effekt im gleichen Gewebe aufwiesen. Weiterhin reichte die ektopische Aktivierung des JAK/STAT-Signalwegs zu diesem späten Entwicklungszeitpunkt aus, um die Mitose zu inhibieren und die Zellen in der Phase G2 des Zellzyklus zu arretieren. Diese Ergebnisse legen den Schluss nahe, dass der JAK/STAT-Signalweg sowohl pro-proliferativ in frühen Flügelscheiben als auch anti-proliferativ zu späten Stadien der Flügelscheiben-Entwicklung wirken kann. Dieser späte anti-proliferative Effekt wurde durch einen nicht-kanonischen Mechanismus der STAT92E-Aktivierung vermittelt, da späte hop defiziente Zellverbände im Vergleich zu Wildtyp-Zellen keine Veränderungen im Ausmaß der zellulären Proliferation aufwiesen. Ferner konnte gezeigt werden, dass eine während der Larvalstadien exprimierte dominant-negative und im N-Terminus deletierte Form von STAT92E (?NSTAT92E) nicht für den anti-proliferativen Effekt verantwortlich ist. Diese Tatsache ist ein weiteres Indiz dafür, dass das vollständige STAT92E den späten anti-proliferativen Effekt verursacht. Um Modulatoren für die von JAK/STAT vermittelte zelluläre Proliferation zu identifieren, wurde ein P-Element-basierter genetischer Interaktions-Screen in einem sensibilisierten genetischen Hintergrund durchgeführt. Insgesamt wurden dazu 2267 unabhängige P-Element-Insertionen auf ihre Wechselwirkung mit der JAK/STAT-Signalaktivität untersucht und 24 interagierende Loci identifiziert. Diese Kandidaten können in folgende Gruppen eingeordnet werden: Zellzyklusproteine, Transkriptionsfaktoren, DNA und RNA bindende Proteine, ein Mikro-RNA-Gen, Komponenten anderer Signaltransduktionswege und Zelladhäsionsproteine. In den meisten Fällen wurden mehrere Allele der interagierenden Kandidatengene getestet. 18 Kandidatengene mit übereinstimmend interagierenden Allelen wurden dann zur weiteren Analyse ausgewählt. Von diesen 18 Kandidaten-Loci wurden 7 mögliche JAK/STAT-Signalwegskomponenten und 6 neue Zielgene des Signalwegs gefunden. Zusammenfassend wurde das Verständnis um STAT92E verbessert. Dieses Protein hat die gleiche Funktion wie das STAT3-Protein der Wirbeltiere und treibt die zelluläre Proliferation voran. Analog zu STAT1 hat STAT92E aber auch einen anti-proliferativen Effekt. Ferner wurden 24 mögliche Modulatoren der JAK/STAT-Signalaktivität identifiziert. Die Charakterisierung dieser Wechselwirkungen eröffnet vielversprechende Wege zu dem Verständnis, wie JAK/STAT die zelluläre Proliferation reguliert und könnte bei der Entwicklung von neuartigen therapeutischen Targets zur Behandlung von Krebskrankheiten und Entwicklungsstörungen beitragen.

Molecular mechanisms controlling the precision of chemokine guided cell migration

By the end of the first day of embryonic development, zebrafish primordial germ cells (PGCs) arrive at the site where the gonad develops. In our study we investigated the mechanisms controlling the precision of primordial germ cell arrival at their target. We found that in contrast with our expectations which were based on findings in Drosophila and mouse, the endoderm does not constitute a preferred migration substrate for the PGCs. Rather, endoderm derivatives are important for later stages of organogenesis keeping the PGC clusters separated. It would be interesting to investigate the precise mechanism by which endoderm controls germ cell position in the gonad. In their migration towards the gonad, zebrafish germ cells follow the gradient of chemokine SDF-1a, which they detect using the receptor CXCR4b that is expressed on their membrane. Here we show that the C-terminal region of CXCR4b is responsible for down-regulation of receptor activity as well as for receptor internalization. We demonstrate that receptor molecules unable to internalize are less potent in guiding germ cells to the site where the gonad develops, thereby implicating chemokine receptor internalization in facilitating precision of migration during chemotaxis in vivo. We demonstrate that while CXCR4b activity positively regulates the duration of the active migration phases, the down-regulation of CXCR4b signalling by internalization limits the duration of this phase. This way, receptor signalling contributes to the persistence of germ cell migration, whereas receptor down-regulation enables the cells to stop and correct their migration path close to the target where germ cells encounter the highest chemokine signal. Chemokine receptors are involved in directing cell migration in different processes such as lymphocyte trafficking, cancer and in the development of the vascular system. The C-terminal domain of many chemokine receptors was shown to be essential for controlling receptor signalling and internalization. It would therefore be important to determine whether the role for receptor internalization in vivo as described here (allowing periodical corrections to the migration route) and the mechanisms involved (reducing the level of signalling) apply for those other events, too.