Transcription factor PU.1 is required for the development of lymphoid and myeloid progenitors during fetal hematopoiesis. By generating chimeric animals using PU.1-/- ES cells or PU.1(-/-) hematopoietic progenitors, we demonstrate that PU.1 functions in an exclusively cell-autonomous manner to regulate the development of the lymphoid-myeloid system. Multipotential lymphoid-myeloid progenitors (AA4.1+, Lin-) are significantly reduced in PU.1(-/-) embryos and fail to differentiate into B lymphoid or myeloid cells in vitro. These results suggest that the lymphoid and myeloid lineages develop in the fetal liver from a common hematopoietic progenitor not shared with erythrocytes and megakaryocytes. Finally, the Ikaros gene is expressed in PU.1 mutant embryos, suggesting that PU.1 and Ikaros are independently required for specification of embryonic lymphoid cell fates.

The ETS-family transcription factor PU.1 is expressed in hematopoietic tissues, with significant levels of expression in the monocytic and B lymphocytic lineages. PU.1 is identical to the Spi-1 proto-oncogene which is associated with the generation of spleen focusforming virus-induced erythroleukemias. An extensive body of in vitro gene regulatory studies has implicated PU.1 as an important, versatile regulator of B lymphoid-and myeloid-specific genes. The first half of the review is designed to coalesce data generated from studies examining the two PU.1 "knockout" animals, which have prompted a reevaluation of the proposed function of PU.1 during hematopoiesis. During hematopoiesis, PU.1 is required for development along the lymphoid and myeloid lineages but needs to be downregulated during erythropoiesis. These unique functional characteristics of PU.1 will be exemplified by contrasting the function of PU.1 with other transcription factors required during fetal hematopoiesis. The second half of this review will reexamine the functional characteristics of PU.1 deduced from traditional biochemical and transactivation assays in light of recent experiments examining the functional behavior of PU.1 in an embryonic stem cell in vitro differentiation system. Working models of how PU.1 regulates promoter and enhancer regions in the B cell and myeloid lineage will be presented and discussed.

Ulcerative colitis (UC) increases the risk of colorectal cancer (CRC), but the mechanisms involved in colitis-to-cancer transition (CCT) are not well understood. CCT may involve a inflammation-dysplasia-carcinoma progression sequence compared to the better characterized adenoma-carcinoma progression sequence associated with sporadic CRC. One common thread may be activating mutations in components of the Wnt/β-catenin signaling pathway, which occur commonly as early events in sporadic CRC. To examine this hypothesis, we evaluated possible associations between Wnt/β-catenin signaling and CCT based on the cancer stem cell (CSC) model. Wnt/β-catenin immunostaining indicated that UC patients have a level of Wnt-pathway-active cells that is intermediate between normal colon and CRC. These UC cells exhibiting activation of the Wnt pathway constituted a major subpopulation (52%+7.21) of the colonic epithelial cells positive for aldehyde dehydrogenase (ALDH), a putative marker of precursor colon CSC (pCCSC). We further fractionated this subpopulation of pCCSC using a Wnt pathway reporter assay. Over successive passages, pCCSCs with the highest Wnt activity exhibited higher clonogenic and tumorigenic potential than pCCSCs with the lowest Wnt activity, thereby establishing the key role of Wnt activity in driving CSC-like properties in these cells. Notably, 5/20 single cell injections of high-Wnt pCCSC resulted in tumor formation, suggesting a correlation with CCT. Attenuation of Wnt/β-catenin in high-Wnt pCCSC by shRNA-mediated downregulation or pharmacological inhibition significantly reduced tumor growth rates. Overall, the results of our study indicates (i) that early activation of Wnt/β-catenin signaling is critical for CCT, and (ii) that high levels of Wnt/β-catenin signaling can further demarcate ALDH+ tumor-initiating cells in the non-dysplastic epithelium of UC patients. As such, our findings offer plausible diagnostic markers and therapeutic target in the Wnt signaling pathway for early intervention in CCT.

The pathogenesis of ulcerative colitis (UC), a major type of inflammatory bowel disease, remains unknown. No model exists that adequately recapitulates the complexity of clinical UC. Here, we take advantage of induced pluripotent stem cells (iPSCs) to develop an induced human UC-derived organoid (iHUCO) model and compared it with the induced human normal organoid model (iHNO). Notably, iHUCOs recapitulated histological and functional features of primary colitic tissues, including the absence of acidic mucus secretion and aberrant adherens junctions in the epithelial barrier both in vitro and in vivo. We demonstrate that the CXCL8/CXCR1 axis was overexpressed in iHUCO but not in iHNO. As proof-of-principle, we show that inhibition of CXCL8 receptor by the small-molecule non-competitive inhibitor repertaxin attenuated the progression of UC phenotypes in vitro and in vivo. This patient-derived organoid model, containing both epithelial and stromal compartments, will generate new insights into the underlying pathogenesis of UC while offering opportunities to tailor interventions to the individual patient.

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Objective. To determine why methotrexate (MTX) exacerbates rheumatoid nodules in some patients, despite the effective suppression of synovial inflammation. Methods. Phorbol myristate acetate (PMA)‐induced differentiation of monocytes into multinucleated giant cells was used as an in vitro model to study the effects of adenosine on nodulosis. Results. MTX at 200‐2,000 nM or the adenosine A1 agonist N5‐cyclopentyl adenosine (CPA) (10−12 to 10‐9 M) or the A2 antagonist 3,7‐dimethyl‐1‐propargylxanthine markedly enhanced giant cell formation, whereas the adenosine A1 antagonist 8‐cyclopentyl‐dipropylxanthine completely reversed these effects. PMA, CPA, and MTX induced adenosine release by cultured monocytes at concentrations consistent with those associated with predominantly A1 effects. Furthermore, surface expression of A1 receptors was found to remain unchanged on the differentiating cells throughout the culture period. Conclusion. Agents that inhibit adenosine A1 receptors might be useful in the treatment of MTX‐induced rheumatoid nodulosis, while still potentiating the A2‐mediated antiinflammatory effects of MTX on synovitis.

In sporadic colon cancer, colon cancer stem cells (CCSCs) initiate tumorigenesis and may contribute to late disease recurrences and metastases. We previously showed that aldehyde dehydrogenase (ALDH) activity (as indicated by the ALDEFLUOR® assay) is an effective marker for highly enriching CCSCs for further evaluation. Here, we used comparative transcriptome and proteome approaches to identify signaling pathways overrepresented in the CCSC population. We found overexpression of several components of the phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway, including PI3KR2, a regulatory subunit of PI3K. LY294002, a PI3K inhibitor, defined the contribution of the PI3K/Akt/mTOR signaling pathway in CCSCs. LY294002-treated CCSCs showed decreases in proliferation, sphere formation and self-renewal, in phosphorylation-dependent activation of Akt, and in expression of cyclin D1. Inhibition of PI3K in vivo reduced tumorigenicity, increased detection of cleaved caspase 3, an indicator of apoptosis, and elevated expression of the inflammatory chemokine, CXCL8. Collectively, these results indicate that PI3K/Akt/mTOR signaling controls CCSC proliferation and CCSC survival, and suggests that it would be useful to develop therapeutic agents that target this signaling pathway.

؊/؊ ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. The PU