Junsuke Uwada

FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), which is essential for phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] production, is an important regulator of lysosomal homeostasis. PIKFYVE dysfunction leads to cytoplasmic vacuolization; however, the underlying mechanism remains unknown. In this study, we explored the cause of vacuole enlargement upon PIKFYVE inhibition in DU145 prostate cancer cells. Enlargement of vacuoles upon PIKFYVE inhibition required glutamine and its metabolism by glutaminases. Addition of ammonia, a metabolite of glutamine, was sufficient to enlarge vacuoles via PIKFYVE inhibition. Moreover, PIKFYVE inhibition led to intracellular ammonium accumulation. Endosome–lysosome permeabilization resulted in ammonium leakage from the cells, indicating ammonium accumulation in the endosomes and lysosomes. Ammonium accumulation and vacuole expansion were suppressed by the lysosomal lumen neutralization. It is therefore assumed that PIKFYVE inhibition interferes with the efflux of NH4+, which formed through protonation of NH3 in the lysosomal lumen, leading to osmotic swelling of vacuoles. Notably, glutamine or ammonium is required for PIKFYVE inhibition-induced suppression of lysosomal function and autophagic flux. In conclusion, this study shows that PIKFYVE inhibition disrupts lysosomal homeostasis via ammonium accumulation.

FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), that is essential for PtdIns(3,5)P ₂ production, is an important regulator of lysosomal homeostasis. PIKFYVE dysfunction leads to cytoplasmic vacuolization; however, the underlying mechanism remains unknown. In this study, we explored the cause of vacuole enlargement upon PIKFYVE inhibition in DU145 prostate cancer cells. Enlargement of vacuoles by PIKFYVE inhibition required glutamine and its metabolism by glutaminases. Addition of ammonia, a metabolite of glutamine, was sufficient to enlarge vacuoles via PIKFYVE inhibition. Moreover, PIKFYVE inhibition led to intracellular ammonium accumulation. Endosome–lysosome permeabilization resulted in ammonium leakage from the cells, indicating ammonium accumulation in the endosomes and lysosomes. Ammonium accumulation and vacuole expansion were suppressed by the lysosomal lumen neutralization. It is therefore assumed that PIKFYVE inhibition interferes with the efflux of NH ₄ ⁺ , which is protonated NH ₃ in the lysosomal lumen, leading to osmotic swelling of vacuoles. Notably, glutamine or ammonium is required for PIKFYVE inhibition-induced suppression of lysosomal function and autophagic flux. In conclusion, this study showed that PIKfyve inhibition disrupts lysosomal homeostasis via ammonium accumulation. Summary statement: Inhibition of the phosphoinositide kinase PIKFYVE results in endosome/lysosome enlargement and impaired lysosomal function. This study showed that the accumulation of glutamine-derived ammonium is the cause of these events.

Inflammatory bowel disease (IBD), which includes Crohn’s disease and ulcerative colitis, is an intestinal disorder that causes prolonged inflammation of the gastrointestinal tract. Currently, the etiology of IBD is not fully understood and treatments are insufficient to completely cure the disease. In addition to absorbing essential nutrients, intestinal epithelial cells prevent the entry of foreign antigens (micro-organisms and undigested food) through mucus secretion and epithelial barrier formation. Disruption of the intestinal epithelial homeostasis exacerbates inflammation. Thus, the maintenance and reinforcement of epithelial function may have therapeutic benefits in the treatment of IBD. Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors for acetylcholine that are expressed in intestinal epithelial cells. Recent studies have revealed the role of mAChRs in the maintenance of intestinal epithelial homeostasis. The importance of non-neuronal acetylcholine in mAChR activation in epithelial cells has also been recognized. This review aimed to summarize recent advances in research on mAChRs for intestinal epithelial homeostasis and the involvement of non-neuronal acetylcholine systems, and highlight their potential as targets for IBD therapy.

17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) converts androstenedione (A4) into testosterone (T), which regulates sex steroid production. Because various mutations of the HSD17B3 gene cause disorder of sex differentiation (DSD) in multiple mammalian species, it is very important to reveal the molecular characteristics of this gene in various species. Here, we revealed the open reading frame of the ovine HSD17B3 gene. Enzymatic activities of ovine HSD17B3 and HSD17B1 for converting A4 to T were detected using ovine androgen receptor-mediated transactivation in reporter assays. Although HSD17B3 also converted estrone to estradiol, this activity was much weaker than those of HSD17B1. Although ovine HSD17B3 has an amino acid sequence that is conserved compared with other mammalian species, it possesses two amino acid substitutions that are consistent with the reported variants of human HSD17B3. Substitutions of these amino acids in ovine HSD17B3 for those in human did not affect the enzymatic activities. However, enzymatic activities declined upon missense mutations of the HSD17B3 gene associated with 46,XY DSD, affecting amino acids that are conserved between these two species. The present study provides basic information and tools to investigate the molecular mechanisms behind DSD not only in ovine, but also in various mammalian species.

Probenecid is a well-known uricosuric agent used for gout treatment through blockage of urate transporter in renal tubules. On the other hand, probenecid can also inhibit multiple channels and transporters including multidrug resistance protein-1 (MRP-1). Therefore, probenecid might support the effect of anti-cancer drugs via inhibition of efflux of these drugs from tumor cells.Conventionally, cancer cell lines have been cultured as two-dimensional (2D) monolayer. However, such condition does not reflect the real tumor situation in vivo. Recently, 3D culture techniques have developed to examine the cancer cell lines with more natural tumor property. In this study, we evaluated the effect of probenecid on prostate cancer cell lines which cultured as multicellular tumor spheroids by culturing in ultra-low attachment plate.Prostate cancer cell line 22Rv1 cultured as spheroid showed lower sensitivity against anti-cancer drug doxorubicin, compared to cells cultured as monolayer. Probenecid treated-spheroid was more sensitive against doxorubicin. Interestingly, we found that probenecid itself has anti-tumor activity in concentration dependent manner. Probenecid was more effective to 3D cultured spheroid than 2D cultured monolayer. In this presentation, we also show the result of other prostate cancer cell lines and discuss the anti-cancer mechanism of probenecid.

Brain acetylcholine is an important neurotransmitter to regulate synaptic transmission in neural circuits and to enhance learning, memory and cognition. Acetylcholine released from nerve terminals acts on muscarinic and nicotinic acetylcholine receptors on plasma membrane followed by the rapid hydrolytic degradation. We found that muscarinic acetylcholine receptors M1 (M1-mAChRs) are highly expressed on intracellular membranes, such as endoplasmic reticulum and Golgi apparatus, in neurons of central nervous system and activate signaling cascades distinct from those of cell surface receptors. The intracellular M1-mAChRs is activated by endogenous acetylcholine following its uptake via a putative transport system and causes cholinergic facilitation of synaptic long-term potentiation in the hippocampus. In addition, the cholinergic synaptic regulation is found to disappear after chronic restraint stress. We herein introduce function of intracellular M1-mAChR in the brain and discuss about possible relation to stress-related neuropsychiatric disorder.