Inhibition of Wnt/β-Catenin Signaling by a Soluble Collagen-Derived Frizzled Domain Interacting with Wnt3a and the Receptors Frizzled 1 and 8

The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth

A System-Wide Investigation of the Dynamics of Wnt Signaling Reveals Novel Phases of Transcriptional Regulation

Aberrant Wnt signaling has been implicated in a wide variety of cancers and many components of the Wnt signaling network have now been identified. Much less is known, however, about how these proteins are coordinately regulated. Here, a broad, quantitative, and dynamic study of Wnt3a-mediated stimulation of HEK 293 cells revealed two phases of transcriptional regulation: an early phase in which signaling antagonists were downregulated, providing positive feedback, and a later phase in which many of these same antagonists were upregulated, attenuating signaling. The dynamic expression profiles of several response genes, including MYC and CTBP1, correlated significantly with proliferation and migration (P<0.05). Additionally, their levels tracked with the tumorigenicity of colon cancer cell lines and they were significantly overexpressed in colorectal adenocarcinomas (P<0.05). Our data highlight CtBP1 as a transcription factor that contributes to positive feedback during the early phases of Wnt signaling and serves as a novel marker for colorectal cancer progression

Myofibroblast-Derived SFRP1 as Potential Inhibitor of Colorectal Carcinoma Field Effect

Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC

Sfrp5 Modulates Both Wnt and BMP Signaling and Regulates Gastrointestinal Organogensis in the Zebrafish, Danio rerio

Sfrp5 belongs to the family of secreted frizzled related proteins (Sfrp), secreted inhibitors of Wingless-MMTV Integration Site (Wnt) signaling, which play an important role in cancer and development. We selected sfrp5 because of its compelling expression profile in the developing endoderm in zebrafish, Danio rerio. In this study, overexpression of sfrp5 in embryos results in defects in both convergent extension (CE) by inhibition of non-canonical Wnt signaling and defects in dorsoventral patterning by inhibition of Tolloid-mediated proteolysis of the BMP inhibitor Chordin. From 25 hours post fertilization (hpf) to 3 days post fertilization (dpf), both overexpression and knockdown of Sfrp5 decrease the size of the endoderm, significantly reducing liver cell number. At 3 dpf, insulin-positive endodermal cells fail to coalesce into a single pancreatic islet. We show that Sfrp5 inhibits both canonical and non-canonical Wnt signaling during embryonic and endodermal development, resulting in endodermal abnormalities. © 2013 Stuckenholz et al

Secreted Frizzled-related protein-1 is a negative regulator of androgen receptor activity in prostate cancer

Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/β-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised β-catenin or β-catenin shRNA, suggesting it does not involve Wnt/β-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca2+ and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of β-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer

Wnt signalling and cancer stem cells

[Abstract] Intracellular signalling mediated by secreted Wnt proteins is essential for the establishment of cell fates and proper tissue patterning during embryo development and for the regulation of tissue homeostasis and stem cell function in adult tissues. Aberrant activation of Wnt signalling pathways has been directly linked to the genesis of different tumours. Here, the components and molecular mechanisms implicated in the transduction of Wnt signal, along with important results supporting a central role for this signalling pathway in stem cell function regulation and carcinogenesis will be briefly reviewed.Ministerio de Ciencia e Innovación; SAF2008-0060

The molecular and cellular signatures of the mouse eminentia thalami support its role as a signalling centre in the developing forebrain

The mammalian eminentia thalami (EmT) (or thalamic eminence) is an embryonic forebrain structure of unknown function. Here, we examined the molecular and cellular properties of the mouse EmT. We first studied mRNA expression of signalling molecules and found that the EmT is a structure, rich in expression of secreted factors, with Wnts being the most abundantly detected. We then examined whether EmT tissue could induce cell fate changes when grafted ectopically. For this, we transplanted EmT tissue from a tau-GFP mouse to the ventral telencephalon of a wild type host, a telencephalic region where Wnt signalling is not normally active but which we showed in culture experiments is competent to respond to Wnts. We observed that the EmT was able to induce in adjacent ventral telencephalic cells ectopic expression of Lef1, a transcriptional activator and a target gene of the Wnt/β-catenin pathway. These Lef1-positive;GFP-negative cells expressed the telencephalic marker Foxg1 but not Ascl1, which is normally expressed by ventral telencephalic cells. These results suggest that the EmT has the capacity to activate Wnt/β-catenin signalling in the ventral telencephalon and to suppress ventral telencephalic gene expression. Altogether, our data support a role of the EmT as a signalling centre in the developing mouse forebrain. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00429-015-1127-3) contains supplementary material, which is available to authorized users

Mikrobiologiczny rozkład siarczanów w ściekach przemysłowych

The study presents comparison of effectiveness in using microbial desulphurisation of dairy industry. Comparative study covers two factors describing performance and effectiveness depending on medium and wastewater content: COD and sulphites. Authoress proves that usage of SRB can be successfully exploited In significant reduction of sulphates and their immediate change into sulphides, which can be separated from the wastewater in form of sludge.W pracy przedstawiono porównanie skuteczności w wykorzystaniu mikroorganizmów do odsiarczania ścieków przemysłu mleczarskiego. Analiza porównawcza obejmuje dwa czynniki opisujące działanie i skuteczność w zależności od medium i zawartości ścieków: ChZT i siarczany. Autorka dowodzi, że użycie BRS może być z powodzeniem wykorzystywane w znacznej redukcji siarczanów i ich natychmiastową zmianę do siarczków, które mogą być oddzielone od ścieków w postaci osadu

Oczyszczanie ścieków z przemysłu mlecznego za pomocą redukcji mikrobiologicznej z użyciem Desulfotomaculum ruminis

Presented results are a continuation of a research programme on use of different types of bacteria in water treatment of industrial wastewater. The general purpose of this study was to check the possibility of using SRB (Sulphate-Reducing Bacteria) for the process of dairy industry wastewater purification. This type of wastewater contain mainly carbohydrates, mainly lactose (30.9%), proteins (23.6%, including 80% of casein) and fats (41.4%) coming from milk and its products [2, 3]. The aim of this study is to investigate the possibility of applying sulphate respiration process to remove organic impurities in case of leakage of industrial wastewater. Conducted research concerns the reduction of sulphate to sulphide in the dairy industry wastewater using Desulfotomaculum ruminis strain of bacteria. Reported study evaluate the effect of reducing the bacterial content of the sulfur in the effluent, depending on the concentration of wastewater. The sulphate breathing process was carried at in laboratory conditions using the SRB on standing culture with modified Starkey medium containing dairy wastewaters as the unique source of carbon and energy. The study examines changes in the COD, level of sulphide content and the pH changes in the test samples. Compared samples contained 30%, 50% and 70% of the dairy effluent from the dairy. The results show the need to find the optimal ratio between the content of the effluent in the suspension being subjected to the SRB, as well as lead the process at the optimum time in order to maintain maximum results. The pH value increased in time. During the first three days the pH value was maintained at a similar level, while in fifth day its value suddenly increased. During the research reaction of tested wastewater samples pH ranged between 6.9-8.0.Zaprezentowane wyniki są kontynuacją program badań na temat użycia różnych typów bakterii w oczyszczaniu ścieków przemysłowych. Głównym celem badań było zbadanie możliwości użycia bakterii redukujących siarczany – SRB w procesie oczyszczania ścieków z przemysłu mlecznego. Ten typ ścieków zawiera głównie węglowodany, głównie laktozę (30,9%), białka (23,6%, w tym 80% kazeiny) oraz tłuszcze (41,4%) pochodzące z mleka i jego produktów [2, 3]. Celem badań jest znalezienie możliwości zastosowania procesu oddychania siarczanów aby usunąć organiczne nieczystości w przypadku wycieku ścieków przemysłowych. Przeprowadzone badania skupiały się na redukcji siarczanów do siarczków w ściekach z przemysłu mleczarskiego przy użyciu szczepu bakterii Desulfotomaculum ruminis. Badania przedstawione w raporcie oceniają efekt redukcji bakteryjnej zawartości siarki w wypływie w zależności od stężenia ścieków. Proces oddychania siarczanów został przeprowadzony w warunkach laboratoryjnych z użyciem SRB na stojącą kulturę bakterii ze zmodyfikowanym nośnikiem Starkeya zawierającym ścieki mleczarskie jako unikalne źródło węgla i energii. W pracy zbadano zmiany w COD, poziom zawartości siarczków i zmiany pH w badanych próbkach. Porównywane próbki zawierały 30%, 50% i 70% ścieku mleczarskiego. Wyniki wskazują na istnienie potrzeby znalezienia optymalnego stosunku między zawartością ścieku w zawiesinie będącej skierowaną do SRB, jak również sposobu przeprowadzania procesu w optymalnym czasie w celu uzyskania maksymalnych wyników. Wartość pH zwiększa się w czasie. Podczas pierwszych trzech dni wartości pH utrzymywały się na podobnym poziomie, podczas gdy piątego dnia wartość nagle wzrosła. Podczas badanej reakcji pH próbek przyjmowało wartości z zakresu 6,9-8,0

Optymalizacja oczyszczania ścieków żywnościowych za pomocą Desulfotomaculum Ruminis w warunkach anaerobowych

One of the most heavily laden pollutants is dairy wastewater with excessive volume of sulphates. Biological methods use sulphate-reducing bacteria Desulfotomaculum ruminis in diary wastewater treatment. Diary wastewater treatment process with the participation of SRB results in secretion of hydrogen sulfide and a decreasing COD, i.e. reducing the concentration of organic substrates. An application of the sulfate reducing bacteria in the treatment of dairy wastewater was investigated. The wastewater impurities formed in the milk and dairy product processing consist of carbohydrates – (mainly lactose), proteins, including important part of casein), and fats. The activity of catabolic growth of the sulfate reducing bacteria cultures was studied. SRB carry out dissimilatory reduction of sulphates, which uses sulphate ions (IV) and (VI) as the final acceptors of electrons and hydrogen. Laboratory studies have shown that sulfides (mostly FeS) precipitated the growth environment during dissimilatory reduction having deterrent effect on the decrease of ions SO42- to S2- In this article there are presented results of treating wastewater containing different concentration of affluent. The sulphate breathing process was carried out in laboratory conditions using the SRB culture on modified Starkey medium containing dairy wastewaters as the unique source of carbon and energy. The study examines and determines the optimum pH level for SRB which ranges between 6.84 to 8.0. There were four samples with different waste water content: 2,5%, 5%, 7,5% and 10%. Dependence of affluent content in waste water was research through measuring the reduction of sulphates to sulfites by increasing the last as well as COD changes.Jednym z największych istniejących zanieczyszczeń wód są ścieki spożywcze zawierająca ogromne ilości siarczanów. Biometody uwzględniają użycie bakterii redukującej siarczany (ang. skrót SRB) Desulfotomaculumruminis w procesach przetwórczych ścieków. Proces przetwórczy ścieków spożywczych z udziałem SRB powoduje wydzielanie siarkowodoru i spadek chemicznego zapotrzebowania na tlen, czyli redukcję stężenia substratów organicznych. Zbadano efekty po dodaniu bakterii redukującej siarczany w procesie przetwórstwa ścieków spożywczych. Zanieczyszczenia wody ściekowej powstałej z mleka i przetworzonych produktów nabiałowych składają się z węglowodorów-(głównie laktozy), protein (w dużej mierze kazein) i tłuszczy. Zbadano również aktywność wzrostu katabolitycznego kultury bakterii redukującej siarczany. SRB przeprowadza dysymilacyjną redukcję siarczanów, które wykorzystują jony siarczanów (IV) oraz (VI), jako główne akceptory elektronów i wodoru. Badania laboratoryjne wykazały, że siarczki (głównie FeS) wzrost osadów w środowisku podczas dysymilacyjnej redukcji, tym samym wstrzymując redukcję jonów z SO42- na S2-. W artykule przedstawiono wyniki przetwórstwa ścieków zawierających różnego rodzaju składniki. W warunkach laboratoryjnych przeprowadzono proces utleniania siarczanowego, przy użyciu kultur bakterii redukującej siarczany na zmodyfikowanym medium Starkey’a zawierającym ścieki spożywcze, które są wyjątkowym źródłem węgla i energii. W pracy sprawdzono i określono optymalny poziom pH dla SRB, które zamknęło się w zakresie od 6,84 do 8,0. Wzięto pod uwagę 4 próbki ze zróżnicowaną zawartością ścieków: 2,5%, 5%, 7,5% oraz 10%. Zależność zawartości istotnych składników w ściekach zbadano przez pomiar redukcji siarczanów do siarczynów i zmniejszenie chemicznego zapotrzebowania na tlen