The pluripotent stem cell-specific transcript ESRG is dispensable for human pluripotency.

Human pluripotent stem cells (PSCs) express human endogenous retrovirus type-H (HERV-H), which exists as more than a thousand copies on the human genome and frequently produces chimeric transcripts as long-non-coding RNAs (lncRNAs) fused with downstream neighbor genes. Previous studies showed that HERV-H expression is required for the maintenance of PSC identity, and aberrant HERV-H expression attenuates neural differentiation potentials, however, little is known about the actual of function of HERV-H. In this study, we focused on ESRG, which is known as a PSC-related HERV-H-driven lncRNA. The global transcriptome data of various tissues and cell lines and quantitative expression analysis of PSCs showed that ESRG expression is much higher than other HERV-Hs and tightly silenced after differentiation. However, the loss of function by the complete excision of the entire ESRG gene body using a CRISPR/Cas9 platform revealed that ESRG is dispensable for the maintenance of the primed and naïve pluripotent states. The loss of ESRG hardly affected the global gene expression of PSCs or the differentiation potential toward trilineage. Differentiated cells derived from ESRG-deficient PSCs retained the potential to be reprogrammed into induced PSCs (iPSCs) by the forced expression of OCT3/4, SOX2, and KLF4. In conclusion, ESRG is dispensable for the maintenance and recapturing of human pluripotency.

Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells.

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

High glucose induces N-linked glycosylation-mediated functional upregulation and overexpression of Cav3.2 T-type calcium channels in neuroendocrine-like differentiated human prostate cancer cells.

Given that Cav3.2 T-type Ca2+ channels were functionally regulated by asparagine (N)-linked glycosylation, we examined effects of high glucose on the function of Cav3.2, known to regulate secretory function, in neuroendocrine-like differentiated prostate cancer LNCaP cells. High glucose accelerated the increased channel function and overexpression of Cav3.2 during neuroendocrine differentiation, the former prevented by enzymatic inhibition of N-glycosylation and cleavage of N-glycans. Hyperglycemia thus appears to induce N-linked glycosylation-mediated functional upregulation and overexpression of Cav3.2 in neuroendocrine-like differentiated prostate cancer cells.

Crystallization and preliminary X-ray crystallographic studies of the axin DIX domain.

Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of beta-catenin by glycogen synthase kinase 3beta. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 91.49, c = 84.92 A. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 A.

Functional upregulation of the H2S/Cav3.2 channel pathway accelerates secretory function in neuroendocrine-differentiated human prostate cancer cells.

Neuroendocrine-differentiated prostate cancer cells may contribute to androgen-independent proliferation of surrounding cells through Ca(2+)-dependent secretion of mitogenic factors. Human prostate cancer LNCaP cells, when neuroendocrine-differentiated, overexpress Cav3.2 T-type Ca(2+) channels that contribute to Ca(2+)-dependent secretion. Given evidence for the acceleration of Cav3.2 activity by hydrogen sulfide (H2S), we examined the roles of the H2S/Cav3.2 pathway and then analyzed the molecular mechanisms of the Cav3.2 overexpression in neuroendocrine-differentiated LNCaP cells. LNCaP cells were differentiated by dibutyryl cyclic AMP. Protein levels and T-type Ca(2+) channel-dependent currents (T-currents) were measured by immunoblotting and whole-cell pacth-clamp technique, respectively. Spontaneous release of prostatic acid phosphatase (PAP) was monitored to evaluate secretory function. The differentiated LNCaP cells exhibited neurite outgrowth, androgen-independent proliferation and upregulation of mitogenic factors, and also showed elevation of Cav3.2 expression or T-currents. Expression of cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS), H2S-forming enzymes, and spontaneous secretion of PAP increased following the differentiation. The augmented T-currents were enhanced by H2S donors and suppressed by inhibitors of CSE, but not CBS. The PAP secretion was reduced by inhibition of CSE or T-type Ca(2+) channels. During differentiation, Egr-1 and REST, positive and negative transcriptional regulators for Cav3.2, were upregulated and downregulated, respectively, and Egr-1 knockdown prevented the Cav3.2 overexpression. Our data suggest that, in neuroendocrine-differentiated LNCaP cells, H2S formed by the upregulated CSE promotes the activity of the upregulated Cav3.2, leading to the elevated secretory functions. The overexpression of Cav3.2 appears to involve upregulation of Egr-1 and downregulation of REST.