Core clock gene BMAL1 and RNA-binding protein MEX3A collaboratively regulate Lgr5 expression in intestinal crypt cells.

The intestinal epithelium is highly regenerative. Rapidly proliferating LGR5+ crypt base columnar (CBC) cells are responsible for epithelial turnover needed to maintain intestinal homeostasis. Upon tissue damage, loss of LGR5+ CBCs can be compensated by activation of quiescent +4 intestinal stem cells (ISCs) or early progenitor cells to restore intestinal regeneration. LGR5+ CBC self-renewal and ISC conversion to LGR5+ cells are regulated by external signals originating from the ISC niche. In contrast, little is known about intrinsic regulatory mechanisms critical for maintenance of LGR5+ CBC homeostasis. We found that LGR5 expression in intestinal crypt cells is controlled by the circadian core clock gene BMAL1 and the BMAL1-regulated RNA-binding protein MEX3A. BMAL1 directly activated transcription of Mex3a. MEX3A in turn bound to and stabilized Lgr5 mRNA. Bmal1 depletion reduced Mex3a and Lgr5 expression and led to increased ferroptosis, which consequently decreased LGR5+ CBC numbers and increased the number of crypt cells expressing +4 ISC marker BMI1. Together, these findings reveal a BMAL1-centered intrinsic regulatory pathway that maintains LGR5 expression in the crypt cells and suggest a potential mechanism contributing to ISC homeostasis.

MEX3A Mediates p53 Degradation to Suppress Ferroptosis and Facilitate Ovarian Cancer Tumorigenesis.

Epithelial ovarian cancer is a highly heterogeneous and malignant female cancer with an overall low survival rate. Mutations in p53 are prevalent in the major ovarian cancer histotype, high-grade serous ovarian carcinoma (HGSOC), while p53 mutations are much less frequent in other ovarian cancer subtypes, particularly in ovarian clear cell carcinoma (OCCC). Advanced stage OCCC with wild-type (WT) p53 has a worse prognosis and increased drug resistance, metastasis, and recurrence than HGSOC. The mechanisms responsible for driving the aggressiveness of WT p53-expressing ovarian cancer remain poorly understood. Here, we found that upregulation of MEX3A, a dual-function protein containing a RING finger domain and an RNA-binding domain, was critical for tumorigenesis in WT p53-expressing ovarian cancer. MEX3A overexpression enhanced the growth and clonogenicity of OCCC cell lines. In contrast, depletion of MEX3A in OCCC cells, as well as ovarian teratocarcinoma cells, reduced cell survival and proliferative ability. MEX3A depletion also inhibited tumor growth and prolonged survival in orthotopic xenograft models. MEX3A depletion did not alter p53 mRNA level but did increase p53 protein stability. MEX3A-mediated p53 protein degradation was crucial to suppress ferroptosis and enhance tumorigenesis. Consistently, p53 knockdown reversed the effects of MEX3A depletion. Together, our observations identified MEX3A as an important oncogenic factor promoting tumorigenesis in ovarian cancer cells expressing WT p53. SIGNIFICANCE: Degradation of p53 mediated by MEX3A drives ovarian cancer growth by circumventing p53 tumor suppressive functions, suggesting targeting MEX3A as a potential strategy for treating of ovarian cancer expressing WT p53.

CXCL14 Maintains hESC Self-Renewal through Binding to IGF-1R and Activation of the IGF-1R Pathway.

Human embryonic stem cells (hESCs) have important roles in regenerative medicine, but only a few studies have investigated the cytokines secreted by hESCs. We screened and identified chemokine (C-X-C motif) ligand 14 (CXCL14), which plays crucial roles in hESC renewal. CXCL14, a C-X-C motif chemokine, is also named as breast and kidney-expressed chemokine (BRAK), B cell and monocyte-activated chemokine (BMAC), and macrophage inflammatory protein-2γ (MIP-2γ). Knockdown of CXCL14 disrupted the hESC self-renewal, changed cell cycle distribution, and further increased the expression levels of mesoderm and endoderm differentiated markers. Interestingly, we demonstrated that CXCL14 is the ligand for the insulin-like growth factor 1 receptor (IGF-1R), and it can activate IGF-1R signal transduction to support hESC renewal. Currently published literature indicates that all receptors in the CXCL family are G protein-coupled receptors (GPCRs). This report is the first to demonstrate that a CXCL protein can bind to and activate a receptor tyrosine kinase (RTK), and also the first to show that IGF-1R has another ligand in addition to IGFs. These findings broaden our understanding of stem cell biology and signal transduction.

Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1-3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting Atf1 hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of Sox2 gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.-Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

CHAC2 is essential for self-renewal and glutathione maintenance in human embryonic stem cells.

Glutathione (GSH), the major non-enzymatic antioxidant, plays a critical role in cellular reactive oxygen species (ROS) neutralization. Moreover, GSH is required for the self-renewal maintenance of human embryonic stem cells (hESCs), and is highly accumulated in undifferentiated cells. Among 8 GSH biosynthesis-related enzymes, we found CHAC2 is highly enriched in undifferentiated hESCs. CHAC2 downregulation in hESCs efficiently decreased the levels of GSH and blocked self-renewal. The self-renewal of sh-CHAC2 cells can be rescued by GSH supplement. CHAC2 downregulation promoted mesoderm differentiation and hampered both teratoma formation and the expression of Nrf2 and glutamate-cysteine ligase (GCL). Notably, CHAC1 knockdown restored the self-renewability of CHAC2-downregulated cells. Although both CHAC1 and CHAC2 purified protein alone showed the catalytic activities to GSH, our data extraordinarily revealed that CHAC2 prevented CHAC1-mediated GSH degradation, which suggests that CHAC2 competes with CHAC1 to maintain GSH homeostasis. This is the first report to demonstrate that CHAC2 is critical for GSH maintenance and the novel roles of the CHAC family in hESC renewal.

Elimination of undifferentiated human embryonic stem cells by cardiac glycosides.

An important safety concern in the use of human pluripotent stem cells (hPSCs) is tumorigenic risk, because these cells can form teratomas after an in vivo injection at ectopic sites. Several thousands of undifferentiated hPSCs are sufficient to induce teratomas in a mouse model. Thus, it is critical to remove all residue-undifferentiated hPSCs that have teratoma potential before the clinical application of hPSC-derived cells. In this study, our data demonstrated the cytotoxic effects of cardiac glycosides, such as digoxin, lanatoside C, bufalin, and proscillaridin A, in human embryonic stem cells (hESCs). This phenomenon was not observed in human bone marrow mesenchymal stem cells (hBMMSCs). Most importantly, digoxin and lanatoside C did not affect the stem cells' differentiation ability. Consistently, the viability of the hESC-derived MSCs, neurons, and endothelium cells was not affected by the digoxin and lanatoside C treatment. Furthermore, the in vivo experiments demonstrated that digoxin and lanatoside C prevented teratoma formation. To the best of our knowledge, this study is the first to describe the cytotoxicity and tumor prevention effects of cardiac glycosides in hESCs. Digoxin and lanatoside C are also the first FDA-approved drugs that demonstrated cytotoxicity in undifferentiated hESCs.

Simple and efficient defect-tailored fiber-based UV-VIS broadband white light generation.

We propose and demonstrate a facile approach for ultraviolet-visible broadband generation from a sapphire crystal core-borosilicate glass cladding hybrid fiber using a laser-heated pedestal growth technique. Considerable formation of F- and F(2)-type color emitters is effectively facilitated by Ti(4+) ions and Al(3+) vacancies, retaining efficient luminescence and high crystallinity of the sapphire core. These color centers intensify the ultraviolet, blue, and green emissions at 370, 450, and 540 nm, whereas the 650-nm red emission is contributed by Cr(3+) in the octahedral sites of the corundum structure. Over 1-mW white light with an optical-to-optical efficiency of up to nearly 5% and 1931 Commission International de l'Eclairage chromaticity coordinate of (0.287, 0.333) is achieved under 325-nm excitation.