The importance of claudin-7 palmitoylation on membrane subdomain localization and metastasis-promoting activities.

BACKGROUND: Claudin-7 (cld7), a tight junction (TJ) component, is also found basolaterally and in the cytoplasm. Basolaterally located cld7 is enriched in glycolipid-enriched membrane domains (GEM), where it associates with EpCAM (EpC). The conditions driving cld7 out of TJ into GEM, which is associated with a striking change in function, were not defined. Thus, we asked whether cld7 serines or palmitoylation affect cld7 location and protein, particularly EpCAM, associations. RESULTS: HEK cells were transfected with EpCAM and wild type cld7 or cld7, where serine phopsphorylation or the palmitoylation sites (AA184, AA186) (cld7(mPalm)) were mutated. Exchange of individual serine phosphorylation sites did not significantly affect the GEM localization and the EpCAM association. Instead, cld7(mPalm) was poorly recruited into GEM. This has consequences on migration and invasiveness as palmitoylated cld7 facilitates integrin and EpCAM recruitment, associates with cytoskeletal linker proteins and cooperates with MMP14, CD147 and TACE, which support motility, matrix degradation and EpCAM cleavage. On the other hand, only cld7(mPalm) associates with TJ proteins. CONCLUSION: Cld7 palmitoylation prohibits TJ integration and fosters GEM recruitment. Via associated molecules, palmitoylated cld7 supports motility and invasion.

The affective storms of school children during night time: do affective dysregulated school children show a specific pattern of sleep disturbances?

In many mentally ill children, a new phenotype of affective and behavioural dysregulation can be observed. Not much is known about development and maintenance of this phenotype. A possible risk factor that has been suggested is disturbed sleep. The co-occurrence of sleep disturbances and psychiatric symptoms is significant and it has been postulated that there are specific patterns of sleep disturbances for different psychiatric symptoms. The aim of this study, therefore, is to investigate the specific relation of sleep disturbances and affective dysregulation in school children and whether a specific pattern of sleep disturbances is associated with or of predictive value for affective dysfunctions. 4,774 school children took part in the Cologne Sleep Study and emotional disturbances and sleep abnormalities were assessed by parent reports. Of the sample, 206 children were identified as showing signs of severe affective and behavioural dysregulation (DP), 276 children were reported as showing a subclinical form (SUB-DP). Both groups reported significantly more sleep disturbances than healthy controls with moderate to large effect sizes. Differences between SUB-DP and DP exist, however, were not of clinical relevance (d < 0.03). Particularly, emotional problems were associated with sleep disturbances with small to moderate correlation coefficients. The regression analysis revealed a small, but significant influence of impaired daytime behaviour on affective dysregulation with 16.1 % of variance being explained. No specific patterns of sleep disturbances could be identified. However, impaired sleep and daytime behaviour in both the SUB-DP and DP group are indicated. Consequently, practitioners should address sleep problems in children with affective dysregulation.

Pancreatic cancer stem cell markers and exosomes - the incentive push.

Pancreatic cancer (PaCa) has the highest death rate and incidence is increasing. Poor prognosis is due to late diagnosis and early metastatic spread, which is ascribed to a minor population of so called cancer stem cells (CSC) within the mass of the primary tumor. CSC are defined by biological features, which they share with adult stem cells like longevity, rare cell division, the capacity for self renewal, differentiation, drug resistance and the requirement for a niche. CSC can also be identified by sets of markers, which for pancreatic CSC (Pa-CSC) include CD44v6, c-Met, Tspan8, alpha6beta4, CXCR4, CD133, EpCAM and claudin7. The functional relevance of CSC markers is still disputed. We hypothesize that Pa-CSC markers play a decisive role in tumor progression. This is fostered by the location in glycolipid-enriched membrane domains, which function as signaling platform and support connectivity of the individual Pa-CSC markers. Outside-in signaling supports apoptosis resistance, stem cell gene expression and tumor suppressor gene repression as well as miRNA transcription and silencing. Pa-CSC markers also contribute to motility and invasiveness. By ligand binding host cells are triggered towards creating a milieu supporting Pa-CSC maintenance. Furthermore, CSC markers contribute to the generation, loading and delivery of exosomes, whereby CSC gain the capacity for a cell-cell contact independent crosstalk with the host and neighboring non-CSC. This allows Pa-CSC exosomes (TEX) to reprogram neighboring non-CSC towards epithelial mesenchymal transition and to stimulate host cells towards preparing a niche for metastasizing tumor cells. Finally, TEX communicate with the matrix to support tumor cell motility, invasion and homing. We will discuss the possibility that CSC markers are the initial trigger for these processes and what is the special contribution of CSC-TEX.

Palmitoylated claudin7 captured in glycolipid-enriched membrane microdomains promotes metastasis via associated transmembrane and cytosolic molecules.

In epithelial cells claudin7 (cld7) is a major component of tight junctions, but is also recovered from glycolipid-enriched membrane microdomains (GEM). In tumor cells, too, cld7 exists in two stages. Only GEM-located cld7, which is palmitoylated, promotes metastasis. Searching for the underlying mechanism(s) revealed the following.The metastatic capacity of the rat pancreatic adenocarcinoma cell line ASML is lost by a knockdown (kd) of cld7 and is not regained by rescuing cld7 with a mutated palmitoylation site (cld7mPalm). ASML-cld7kd and ASML-cld7mPalm cells show reduced motility and invasiveness. This is due to cld7, but not cld7mPalm associating with α6ß4, ezrin, uPAR and MMP14, which jointly support motility and invasion. Palmitoylated cld7 also is engaged in drug resistance by repressing Pten, allowing activation of the antiapoptotic PI3K/Akt pathway. An association of cld7mPalm with the major Pten phosphorylating kinases does not restore apoptosis resistance as phosphorylated Pten is not guided towards GEM to compete with non-phosphorylated Pten. The pathway whereby palmitoylated cld7 supports expression of several EMT genes and nuclear translocation of EMT transcription factors remains to be unraveled. An association with Notch, reduced in ASML-cld7mPalm cells, might be the starting point. Finally, GEM-located, palmitoylated cld7 associates with several components of vesicle transport machineries engaged in exosome biogenesis.Taken together, prerequisites for cld7 acting as a cancer-initiating cell marker are GEM location and palmitoylation, which support a multitude of associations and integration into exosomes. The latter suggests palmitoylated cld7 contributing to message transfer via exosomes.

Claudin-7 promotes the epithelial-mesenchymal transition in human colorectal cancer.

In colorectal cancer (CoCa) EpCAM is frequently associated with claudin-7. There is evidence that tumor-promoting EpCAM activities are modulated by the association with claudin-7. To support this hypothesis, claudin-7 was knocked-down (kd) in HT29 and SW948 cells. HT29-cld7kd and SW948-cld7kd cells display decreased anchorage-independent growth and the capacity for holoclone-, respectively, sphere-formation is reduced. Tumor growth is delayed and cld7kd cells poorly metastasize. In line with this, migratory and invasive potential of cld7kd clones is strongly impaired, migration being inhibited by anti-CD49c, but not anti-EpCAM, although motility is reduced in EpCAM siRNA-treated cells. This is due to claudin-7 recruiting EpCAM in glycolipid-enriched membrane fractions towards claudin-7-associated TACE and presenilin2, which cleave EpCAM. The cleaved intracellular domain, EpIC, promotes epithelial-mesenchymal transition (EMT)-associated transcription factor expression, which together with fibronectin and vimentin are reduced in claudin-7kd cells. But, uptake of HT29wt and SW948wt exosomes by the claudin-7kd lines sufficed for transcription factor upregulation and for restoring motility. Thus, claudin-7 contributes to motility and invasion and is required for recruiting EpCAM towards TACE/presenilin2. EpIC generation further supports motility by promoting a shift towards EMT. Notably, EMT features of cld7-competent metastatic CoCa cells can be transferred via exosomes to poorly metastatic cells.

Severe mood dysregulation: in the "light" of circadian functioning.

Severe affective and behavioral dysregulation, labeled as severe mood dysregulation (SMD), is a widely spread phenomenon among adolescent psychiatric patients. This phenotype constitutes severe impairment across multiple settings, including various symptoms, such as non-episodic anger, mood instability, and hyperarousal. Moreover, SMD patients often show depression and reduced need for sleep. Despite a lifetime prevalence of 3.3%, systematic research is still scarce, and treatments that have been established do not account for the range of symptoms present in SMD. Considering the circadian dysfunctions, two hormones, melatonin and cortisol, are essential. When these hormones are dysregulated, the circadian rhythm gets out of synchrony. Since evidence is emerging showing that the worse the sleep-wake cycle is entrained, the worse the psychiatric symptoms are depicted, the importance of proper circadian functioning becomes clear. Chronotherapy as the controlled exposure to environmental stimuli (e.g. light) acting on biological rhythms has shown therapeutic effects. In both seasonal and major depression chronotherapy has been implemented, decreasing depressive symptoms and stabilizing circadian rhythms. Preliminary evidence from SMD related disorders, namely attention-deficit/hyperactivity disorder and pediatric bipolar depression, indicates that morning light therapy elicits positive influences on other symptoms as well. Hence, light therapy might not only be effective for depressive symptoms and circadian rhythms, but might also be beneficial for symptoms including inattention and irritability. We hypothesize that light therapy might be a helpful adjunctive treatment enhancing affective and circadian functioning, and eliciting positive influences on behavior. Physiologically, changes of both cortisol levels and melatonin production are expected.