WNT signalling in the normal human adult testis and in male germ cell neoplasms

2020 ◽  
Vol 35 (9) ◽  
pp. 1991-2003
Author(s):  
Julia C Young ◽  
Genevieve Kerr ◽  
Diana Micati ◽  
John E Nielsen ◽  
Ewa Rajpert-De Meyts ◽  
...  
Keyword(s):  
Germ Cell ◽  
Large Scale ◽  
Wnt Pathway ◽  
Wnt Signalling ◽  
Human Testis ◽  
Beta Catenin ◽  
Pathway Function ◽  
Normal Spermatogenesis ◽  
And Migration ◽  
Canonical Wnt

Abstract STUDY QUESTION Is WNT signalling functional in normal and/or neoplastic human male germ cells? SUMMARY ANSWER Regulated WNT signalling component synthesis in human testes indicates that WNT pathway function changes during normal spermatogenesis and is active in testicular germ cell tumours (TGCTs), and that WNT pathway blockade may restrict seminoma growth and migration. WHAT IS KNOWN ALREADY Regulated WNT signalling governs many developmental processes, including those affecting male fertility during early germ cell development at embryonic and adult (spermatogonial) ages in mice. In addition, although many cancers arise from WNT signalling alterations, the functional relevance and WNT pathway components in TGCT, including germ cell neoplasia in situ (GCNIS), are unknown. STUDY DESIGN, SIZE, DURATION The cellular distribution of transcripts and proteins in WNT signalling pathways was assessed in fixed human testis sections with normal spermatogenesis, GCNIS and seminoma (2–16 individuals per condition). Short-term (1–7 h) ligand activation and long-term (1–5 days) functional outcomes were examined using the well-characterised seminoma cell line, TCam-2. Pathway inhibition used siRNA or chemical exposures over 5 days to assess survival and migration. PARTICIPANTS/MATERIALS, SETTING, METHODS The cellular localisation of WNT signalling components was determined using in situ hybridisation and immunohistochemistry on Bouin’s- and formalin-fixed human testis sections with complete spermatogenesis or germ cell neoplasia, and was also assessed in TCam-2 cells. Pathway function tests included exposure of TCam-2 cells to ligands, small molecules and siRNAs. Outcomes were measured by monitoring beta-catenin (CTNNB1) intracellular localisation, cell counting and gap closure measurements. MAIN RESULTS AND THE ROLE OF CHANCE Detection of nuclear-localised beta-catenin (CTNNB1), and key WNT signalling components (including WNT3A, AXIN2, TCF7L1 and TCF7L2) indicate dynamic and cell-specific pathway activity in the adult human testis. Their presence in germ cell neoplasia and functional analyses in TCam-2 cells indicate roles for active canonical WNT signalling in TGCT relating to viability and migration. All data were analysed to determine statistical significance. LARGE SCALE DATA No large-scale datasets were generated in this study. LIMITATIONS, REASONS FOR CAUTION As TGCTs are rare and morphologically heterogeneous, functional studies in primary cancer cells were not performed. Functional analysis was performed with the only well-characterised, widely accepted seminoma-derived cell line. WIDER IMPLICATIONS OF THE FINDINGS This study demonstrated the potential sites and involvement of the WNT pathway in human spermatogenesis, revealing similarities with murine testis that suggest the potential for functional conservation during normal spermatogenesis. Evidence that inhibition of canonical WNT signalling leads to loss of viability and migratory activity in seminoma cells suggests that potential treatments using small molecule or siRNA inhibitors may be suitable for patients with metastatic TGCTs. STUDY FUNDING AND COMPETING INTEREST(S) This study was funded by National Health and Medical Research Council of Australia (Project ID 1011340 to K.L.L. and H.E.A., and Fellowship ID 1079646 to K.L.L.) and supported by the Victorian Government’s Operational Infrastructure Support Program. None of the authors have any competing interests.

scholarly journals Leptin regulates glucose homeostasis via the canonical WNT pathway

2021 ◽  
Author(s):  
Kaj Kamstra ◽  
Mohammed Z. Rizwan ◽  
Julia A. Horsfield ◽  
Dominik Pretz ◽  
Peter R. Shepherd ◽  
...  
Keyword(s):  
Body Weight ◽  
Glucose Homeostasis ◽  
Wnt Pathway ◽  
Wnt Signalling ◽  
Beta Catenin ◽  
Mediobasal Hypothalamus ◽  
Adult Mice ◽  
Conditional Deletion ◽  
Canonical Wnt Pathway ◽  
Canonical Wnt

SummaryLeptin is a body weight regulatory hormone, but it is arguably even more potent at regulating blood glucose levels. To further our understanding of the molecular mechanisms by which leptin controls glucose homeostasis, we have used transgenic zebrafish models and conditional deletion of beta catenin in the mediobasal hypothalamus of adult mice to show that Wnt signalling in the brain mediates glucoregulatory effects of leptin. In zebrafish, under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis. In times of nutrient excess, we found that leptin also regulates body weight and size in this species. Using a Wnt signalling reporter fish, we show that leptin directly activates the canonical Wnt pathway in vivo. Pharmacological inhibition of this pathway prevented the leptin-induced improvement in glucose tolerance. In adult mice, conditional deletion of the key Wnt effector molecule, β-catenin, in the mediobasal hypothalamus of male mice confirmed the essential role of the Wnt pathway in mediating leptin action and the neuroendocrine regulation of glucose homeostasis. Adult-onset β-catenin deletion in the mediobasal hypothalamus led to glucose intolerance, exacerbation of caloric intake and body weight gain under high fat diet, as well as resistance to exogenous leptin.

C. elegans POP-1/TCF functions in a canonical Wnt pathway that controls cell migration and in a noncanonical Wnt pathway that controls cell polarity

Development ◽  
2001 ◽  
Vol 128 (4) ◽  
pp. 581-590 ◽  
Author(s):  
M. Herman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Polarity ◽  
Wnt Pathway ◽  
Beta Catenin ◽  
Wild Type ◽  
C Elegans ◽  
Cell Divisions ◽  
Canonical Wnt Pathway ◽  
Canonical Wnt

In Caenorhabditis elegans, Wnt signaling pathways are important in controlling cell polarity and cell migrations. In the embryo, a novel Wnt pathway functions through a (beta)-catenin homolog, WRM-1, to downregulate the levels of POP-1/Tcf in the posterior daughter of the EMS blastomere. The level of POP-1 is also lower in the posterior daughters of many anteroposterior asymmetric cell divisions during development. I have found that this is the case for of a pair of postembryonic blast cells in the tail. In wild-type animals, the level of POP-1 is lower in the posterior daughters of the two T cells, TL and TR. Furthermore, in lin-44/Wnt mutants, in which the polarities of the T cell divisions are frequently reversed, the level of POP-1 is frequently lower in the anterior daughters of the T cells. I have used a novel RNA-mediated interference technique to interfere specifically with pop-1 zygotic function and have determined that pop-1 is required for wild-type T cell polarity. Surprisingly, none of the three C. elegans (beta)-catenin homologs appeared to function with POP-1 to control T cell polarity. Wnt signaling by EGL-20/Wnt controls the migration of the descendants of the QL neuroblast by regulating the expression the Hox gene mab-5. Interfering with pop-1 zygotic function caused defects in the migration of the QL descendants that mimicked the defects in egl-20/Wnt mutants and blocked the expression of mab-5. This suggests that POP-1 functions in the canonical Wnt pathway to control QL descendant migration and in novel Wnt pathways to control EMS and T cell polarities.

scholarly journals Is resistant starch protective against colorectal cancer via modulation of the WNT signalling pathway?

2015 ◽  
Vol 74 (3) ◽  
pp. 282-291 ◽  
Author(s):  
Fiona C. Malcomson ◽  
Naomi D. Willis ◽  
John C. Mathers
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Resistant Starch ◽  
Wnt Pathway ◽  
Wnt Signalling ◽  
Signalling Pathway ◽  
Wnt Signalling Pathway ◽  
Crypt Cell Proliferation ◽  
And Migration ◽  
Epigenetic Modulation

Epidemiological and experimental evidence suggests that non-digestible carbohydrates (NDC) including resistant starch are protective against colorectal cancer. These anti-neoplastic effects are presumed to result from the production of the SCFA, butyrate, by colonic fermentation, which binds to the G-protein-coupled receptor GPR43 to regulate inflammation and other cancer-related processes. The WNT pathway is central to the maintenance of homeostasis within the large bowel through regulation of processes such as cell proliferation and migration and is frequently aberrantly hyperactivated in colorectal cancers. Abnormal WNT signalling can lead to irregular crypt cell proliferation that favours a hyperproliferative state. Butyrate has been shown to modulate the WNT pathway positively, affecting functional outcomes such as apoptosis and proliferation. Butyrate's ability to regulate gene expression results from epigenetic mechanisms, including its role as a histone deacetylase inhibitor and through modulating DNA methylation and the expression of microRNA. We conclude that genetic and epigenetic modulation of the WNT signalling pathway may be an important mechanism through which butyrate from fermentation of resistant starch and other NDC exert their chemoprotective effects.

Alterations In Wnt Signalling In the Megakaryocytic Lineage Leads to Bone Marrow Failure and Myelofibrosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 628-628
Author(s):  
Simon D Calaminus ◽  
Gareth Inman ◽  
Cedric Ghaevert ◽  
Owen Sansom ◽  
Steve P Watson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Wnt Pathway ◽  
Bone Marrow Failure ◽  
Prognostic Score ◽  
Fold Increase ◽  
Wnt Signalling ◽  
Beta Catenin ◽  
Bone Marrow Fibrosis ◽  
Platelet Factor ◽  
Marrow Fibrosis

Abstract Abstract 628 Introduction: Wnt signalling is fundamental in controlling stem cell self-renewal, cell proliferation and development in multicellular organisms. Stabilization of beta catenin or loss of the scaffold protein adenomatis polyposis coli (APC) causes aberrant activation of wnt signalling and often leads to cancer. Mutations to wnt pathway members in haematopoietic stem cells leads to haematopoietic failure and rapid lethality. In this study, we demonstrate that aberrant wnt signalling in the megakaryocyte lineage underlies myelofibrosis. Methods: We created a series of mice with altered wnt pathway signalling in their megakaryocytic lineage using PF4-Cre (platelet factor 4 cre) as follows: Ctnnb1fx(ex3)/wt_ Expresses stabilized active beta catenin henceforth termed PF4bcat+; APCfx/fx_ Loss of APC stabilises beta catenin termed PF4APC-KO; Ctnnb1fx/fx b-catenin knockout termed PF4bcat-KO. Results: By day 40, PF4bcat+ and PF4APC-KO mice are severely underweight, anaemic (wt 6.8+0.2×106/ml v. PF4bcat+ 4.2+0.3×106/ml, PF4APC-KO 5.5+0.5×106/ml), and have a significant reduction in platelet number (wt 1033+37×106/ml v. PF4bcat+ 717+57×106/ml, PF4APC-KO 747+68×106/ml). Furthermore PF4bcat+ and PF4APC-KO mice develop bone marrow fibrosis and consistently die within 50 days of birth. Both populations of mice have splenic extramedullary haemopoiesis with hyperplasia of splenic megakaryocytes, leading to a dramatic increase in spleen to body size ratio. In addition, both PF4bcat+ and PF4APC-KO mice have increased peripheral blood levels of active TGFb, providing a likely molecular basis of the induction in bone marrow fibrosis. PF4APC-KO and PF4bcat+ mutant mice show a dramatic (>10-fold) increase in platelet b-catenin protein levels over wt samples. By comparison, human myelofibrosis patients (n=16) show a 2.7+0.6-fold increase in platelet b-catenin expression over controls. Moreover, overexpression of b-catenin within human patient samples is correlated with a worsening Primary Myelofibrosis prognostic score (Blood, 2009 vol 113 p. 2895), with those patients with a low score (n=7) having a 1.3+/−0.37-fold increase over control, intermediate score (n=4) 4.52+/−1.23-fold, and high score (n=1) 4.56-fold. In contrast PF4bcat-KO mice show no changes in whole blood counts, weight, or evidence of splenic extramedullary haematopoiesis, indicating that b-catenin removal does not adversely affect megakaryocyte development or function. Conclusions: Stabilisation of b-catenin within mouse megakaryocytes leads to a myelodysplastic disorder and myelofibrosis. This finding demonstrates a defined role for aberrant activation of the wnt signalling pathway and marks the wnt pathway and the megakaryocyte lineage as important potential drug targets for the treatment of myelodysplastic disorders. Disclosures: No relevant conflicts of interest to declare.

Noncanonical Wnt5a Is Induced by HTLV-1 bZIP Factor, and Supports Proliferation and Migration of Adult T-Cell Leukemia Cell.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2405-2405
Author(s):  
Jun-ichirou Yasunaga ◽  
Guangyong Ma ◽  
Jun Fan ◽  
Masao Matsuoka
Keyword(s):  
T Cell ◽  
Wnt Signaling ◽  
Wnt Pathway ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Adult T Cell Leukemia ◽  
Canonical Wnt Pathway ◽  
Bzip Factor ◽  
And Migration ◽  
Canonical Wnt

Abstract Abstract 2405 Adult T-cell leukemia (ATL) is a fatal malignancy of CD4-positive T-cells caused by human T-cell leukemia virus type1 (HTLV-1). HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1 provirus, and is consistently expressed in HTLV-1-infected cells. It has been shown that HBZ promotes proliferation of T-cells, and HBZ-expressing transgenic mice develop T-cell lymphomas and systemic inflammations, suggesting that HBZ has the critical roles in pathogenesis of HTLV-1. However, the molecular mechanisms of HBZ-induced oncogenesis have not been clarified yet. The Wnt signaling is a highly conserved cellular signaling pathway in eukaryotes and the Wnt ligands have been found to trigger multiple pathways of Wnt signaling. The most well-studied cascade is the Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is β-catenin-dependent and mainly controls cell differentiation, proliferation, and apoptosis. Aberrant activation of the canonical Wnt pathway has been linked to many cancers. In contrast, it is known that the noncanonical Wnt pathway antagonizes the canonical pathway, while a representative noncanonical Wnt ligand, Wnt5a, is suggested to have both oncogenic and tumor suppressive properties in several malignancies. The significance of those Wnt pathways in HTLV-1-associated oncogenesis is still obscure. In this study, we found that HBZ markedly suppressed the canonical Wnt/β-catenin pathway. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets LEF1 and TCF1, the key transcription factors of the pathway, and impairs its DNA-binding ability. In contrast, HTLV-1 Tax protein interacted with Dvl and DAPLE, which are the regulators of the canonical Wnt signaling, and activated the pathway. Indeed, the canonical Wnt activation is not observed in ATL cells, and enforced activation of β-catenin in ATL cells inhibited cellular proliferation, implying that activation of the canonical Wnt pathway might have suppressive effect on ATL cells. On the other hand, HBZ induces the transcription of the noncanonical Wnt5a by enhancing its promoter activity. In addition, Wnt5a transcription level in ATL cells is significantly higher than in HTLV-1-uninifected control cells. Knockdown of Wnt5a impairs both proliferation and migration of ATL cells, suggesting that HBZ-induced Wnt5a supports development and progression of ATL. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Canonical Wnt signaling regulates branching morphogenesis of submandibular gland by modulating levels of lama5

2021 ◽  
Vol 52 ◽  
Author(s):  
Liming Gou ◽  
Xiaochun Ren ◽  
Ping Ji
Keyword(s):  
Organ Culture ◽  
Signaling Pathway ◽  
Branching Process ◽  
Wnt Pathway ◽  
Branching Morphogenesis ◽  
Beta Catenin ◽  
Fgf Signaling ◽  
Canonical Wnt Pathway ◽  
Culture Model ◽  
Canonical Wnt

Background: Branching morphogenesis is a crucial developmental mechanism for the formation of a typical bush-like structure of the submandibular gland (SMG). However, the detailed mechanism underlying this process remains to be fully understood. Here, we investigate whether a cross-talking may exist between Wnt/beta-catenin signaling pathway and lama5 during the branching process in SMG development. Methods: Embryonic mouse SMG organ culture model was established, and the validity of this model was confirmed. The roles of Wnt/beta-catenin signaling pathway, FGF signaling, and Lama5 in the branching process were investigated by morphogenesis assays. And the interactions between these signaling were also investigated and demonstrated by morphogenesis assays and gene expression patterns. Results: We demonstrated that E12 or E13 SMG organ culture model can be used as an ideal approach to study the process of branching morphogenesis. And branching morphogenesis assay revealed that the epithelial branching process would be promoted when the canonical Wnt pathway was inhibited and be significantly suppressed when wnt pathway is over activated. Further experiments indicated that FGF signaling acts most likely acts upstream as a negative regulator of the canonical Wnt pathway during the branching process, whose effect could be partially reversed by Wnt3a. And we further demonstrated that Wnt/beta-catenin signaling regulates the branching morphogenesis through Lama5. Conclusion: Our present work demonstrated that Wnt/beta-catenin signaling pathway acting downstream of FGF signaling may serve as a negative regulatory mechanism in the process of SMG branching morphogenesis through Lama5.

scholarly journals Expression patterns of HENMT1 and PIWIL1 in human testis: implications for transposon expression

Reproduction ◽  
2017 ◽  
Vol 154 (4) ◽  
pp. 363-374 ◽  
Author(s):  
A L Hempfling ◽  
S L Lim ◽  
D L Adelson ◽  
J Evans ◽  
A E O’Connor ◽  
...  
Keyword(s):  
Germ Cell ◽  
Expression Patterns ◽  
Human Testis ◽  
Testicular Germ Cell ◽  
Cell Content ◽  
Testicular Germ Cell Tumours ◽  
Normal Spermatogenesis ◽  
Low Levels ◽  
Pachytene Spermatocytes ◽  
Pirna Pathway

This study aimed to define the expression patterns of HENMT1 and PIWI proteins in human testis and investigate their association with transposon expression, infertility sub-type or development of testicular germ cell tumours (TGCTs). Testis biopsies showing normal spermatogenesis were used to identify normal localisation patterns of HENMT1 and PIWIL1 by immunolocalisation and RT-PCR after laser microdissection. 222 testis biopsies representing normal spermatogenesis, hypospermatogenesis, spermatogenic arrests, Sertoli cell-only (SCO) tumours and TGCTs were analysed by RT-qPCR for expression ofHENMT1/PIWIL1/PIWIL2/PIWIL3/PIWIL4andLINE-1. Additionally,HENMT1-overexpressing TCam2 seminoma cell lines were analysed for the same parameters by RT-qPCR. We found thatHENMT1andPIWIL1are coexpressed in pachytene spermatocytes and spermatids. Expression ofHENMT1,PIWIL1andPIWIL2was mainly dependent on germ cell content but low levels of expression were also detected in some SCO samples. Levels ofHENMT1,PIWIL1andPIWIL2expression were low in TGCT. Samples withHENMT1, PIWIL2andPIWIL4expression showed significantly (P < 0.05) lower transposon expression compared to samples without expression in the same histological group. HENMT1-overexpressing TCam2 cells showed lowerLINE-1expression than empty vector-transfected control lines. Our findings support that the transposon-regulating function of the piRNA pathway found in the mouse is conserved in adult human testis. HENMT1 and PIWI proteins are expressed in a germ-cell-specific manner and required for transposon control.

Molecular Docking Studies to Evaluate Small Molecule Inhibitors of Wnt/Betacatenin Signaling Pathway

2021 ◽  
Vol 11 (5) ◽  
Author(s):  
Sankari Dantu Sai Shyama Lakshmi ◽  
Maka Sai Sailaja ◽  
Dalal Swetha ◽  
Chanda Chandrasekhar ◽  
Aluru Ranganadha Reddy
Keyword(s):  
Hydrogen Bond ◽  
Molecular Docking ◽  
Hydrogen Bonds ◽  
Wnt Pathway ◽  
Docking Studies ◽  
Wnt Signalling ◽  
Signalling Pathway ◽  
Wnt Signalling Pathway ◽  
Canonical Wnt Pathway ◽  
Canonical Wnt

Canonical Wnt pathway or β catenin dependent pathway is one of the highly conserved signalling pathway which can control gene expression and regulate cell proliferation, cell adhesion, cell migration, cell polarity and organogenesis. Abnormal regulation of β catenin in the canonical wnt signalling pathway leads to transcription of several genes involved in oncogenic programs. Aberrant signalling of the canonical wnt pathway was observed in several types of cancers including hepatocarcinoma, colorectal cancer and lung cancer. Many small molecules were observed to have the potential to block the aberrant wnt signalling pathway by allosteric binding and inhibiting β catenin molecule. The current study involves screening for ligands which can have strong allosteric binds to β catenin and inhibit wnt signalling pathway. Molecular docking studies were used to evaluate the binding capacity of the selected ligands. Curcumin, Cardamonin, FH535 and ICRT-3 were used as ligands for the molecular docking study with β catenin binding Transcription factor -4 receptor. All chosen ligands have exhibited significant binding energies with the receptor. The highest -9.518272 kcal/mol with Cardamonin followed by -9.28359 kcal/mol with FH535, -8.422604 kcal/mol with curcumin and the least -8.407231 kcal/mol with ICRT-3. All the ligands showed at least 1 hydrogen bond with the target receptor whereas Cardamonin showed 3 hydrogen bonds. Curcumin is a close second forming 2 hydrogen bonds while FH535 and ICRT-3 form only 1 hydrogen bond. The 2D interactions of the ligand and the molecule are visualised by using chimera. We observed Cardamonin to have a very strong binding affinity with the target receptor. Cardamonin can be a suitable drug candidate and might have the potential to inhibit the β catenin dependent wnt signalling pathway.

Influence of PI-3K/Akt Pathway on Wnt Signalling in Myeloid Progenitor Cells. Evidence for a Role of Autocrine/Paracrine Wnt Regulation in CFU-GM Proliferation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2892-2892
Author(s):  
Georgios Nteliopoulos ◽  
Stephen B Marley ◽  
Myrtle Y Gordon
Keyword(s):  
Conditioned Medium ◽  
Progenitor Cell ◽  
Wnt Pathway ◽  
Wnt Signalling ◽  
Myeloid Progenitor ◽  
Wnt Proteins ◽  
Cd34 Cells ◽  
Myeloid Progenitor Cell ◽  
Progenitor Cell Proliferation ◽  
Canonical Wnt

Abstract We have used a colony replating assay in conjunction with manipulation of the Wnt and PI-3K pathways to investigate regulation of myeloid progenitor cell proliferation/differentiation. We found that PI-3 kinase pathway via Akt acts as a proliferative brake and promotes differentiation in IL3-driven myelopoiesis, since inhibition of PI-3K/Akt pathway with LY294002 (PI-3 kinase inhibitor) and SH-5 (Akt inhibitor) increased proliferation (reduced differentiation). We investigated the involvement of Wnt signalling in CFU-GM proliferation by using exogenous recombinant canonical Wnt3a and non-canonical Wnt5a. We showed that both of the Wnt members cannot support colony growth alone, but when added to IL3 they increased proliferation potential compared with the IL3 control, indicating an involvement of canonical Wnt/β-catenin and non-canonical Wnt pathways in the myeloid progenitor cell proliferation. Immunoblotting analysis indicated that Wnt5a acts independently of β-catenin. Dkk-1 (Wnt-pathway inhibitor) alone did not affect IL-3 dependent proliferation but when combined with recombinant Wnts as expected it abrogated the effects of Wnt3a but not Wnt5a (acts as canonical-Wnt inhibitor). This was confirmed by β-catenin protein levels. Surprisingly, when Dkk-1 was added to LY294002 or SH-5, it blocked their proliferative effects. Dkk-1 acts as functional Wnt-receptor disabler and the finding that it blocks proliferation induced by PI-3K/Akt inhibitors’ indicates a link between the PI-3K and the Wnt signalling pathways. We hypothesised that increased proliferation induced by LY294002 or SH-5 was not mediated by downstream activation of the Wnt pathway but by induced endogenous expression of Wnt proteins and activation of the surface receptor. We conclude that there is a production of endogenous Wnt proteins that increases proliferation. Endogenous Wnt production has been reported in primitive haematopoietic cells so there is potential for a paracrine or autocrine role for these cell regulators. We tested this hypothesis in CD34+ cells and found the addition of SH-5 to IL3 creates an autocrine loop of endogenous Wnt production, which results in the phosphorylation and activation of the LRP6 receptor and the initiation of the canonical signalling pathway. Furthermore, the conditioned medium of cultured CD34+ cells was concentrated by using filtration devices (30kD cut-off) and added to IL3 to support the growth of progenitors of another sample in a CFU-GM assay. We indicated that Wnt production and secretion can act in a paracrine way as well, since IL3+SH-5 conditioned medium increased the proliferative index of CFU-GM cells whereas IL3 conditioned medium did not have significant effect. Dkk-1 abrogated the IL3+SH-5 conditioned medium’s induced proliferation, suggesting that the growth factors that had the proliferative effects were Wnt members. In conclusion, our data suggest that IL3 via PI-3K pathway promotes differentiation of progenitor myeloid cells through inhibition of endogenous Wnt production.

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