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Male penis is required to become erect during copulation. In the upper (dorsal) part of penis, the erectile tissue termed corpus cavernosum (CC) plays fundamental roles for erection by regulating the inner blood flow. When blood flows into the CC, the microvascular complex termed sinusoidal space is reported to expand during erection. A novel in vitro explant system to analyze the dynamic erectile responses during contraction/relaxation is established. The current data show regulatory contraction/relaxation processes induced by phenylephrine (PE) and nitric oxide (NO) donor mimicking dynamic erectile responses by in vitro CC explants. Two-photon excitation microscopy (TPEM) observation shows the synchronous movement of sinusoidal space and the entire CC. By taking advantages of the CC explant system, tadalafil (Cialis) was shown to increase sinusoidal relaxation. Histopathological changes have been generally reported associating with erection in several pathological conditions. Various stressed statuses have been suggested to occur in the erectile responses by previous studies. The current CC explant model enables to analyze such conditions through directly manipulating CC in the repeated contraction/relaxation processes. Expression of oxidative stress marker and contraction-related genes, Hypoxia-inducible factor 1-alpha (Hif1a), glutathione peroxidase 1 (Gpx1), Ras homolog family member A (RhoA), and Rho-associated protein kinase (Rock), was significantly increased in such repeated contraction/relaxation. Altogether, it is suggested that the system is valuable for analyzing structural changes and physiological responses to several regulators in the field of penile medicine.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) specifically catalyzes the transfer of arachidonoyl-CoA to lysophosphoinositides. LPIAT-/- mice have been shown to have severe defects in the brain and liver; however, the exact molecular mechanisms behind these conditions are not well understood. As immune cells have been implicated in liver inflammation based on disfunction of LPIAT1, we generated Raw264.7 macrophages deficient in LPIAT1, using shRNA and CRISPR/Cas9. The amount of C38:4 species in phosphoinositides, especially in PtdInsP2 , was remarkably decreased in these cells. Unlike in wild-type cells, LPIAT1-deficient cells showed prolonged oscillations of intracellular Ca2+ upon UDP stimulation, which is known to activate phospholipase Cβ through the Gq-coupled P2Y6 receptor, even in the absence of extracellular Ca2+ . It is speculated that the prolonged Ca2+ response may be relevant to the increased risk of liver inflammation induced by LPIAT1 disfunction.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P(2). Sperm from VSP(−/−) mice show more Ca(2+) influx upon capacitation than VSP(+/−) mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P(2)-sensitive K(+) channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca(2+) influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P(2) in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P(2) distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P(2) distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P(2) distribution plays important roles in ion channel regulation as well as sperm motility.

Macrophages endocytose modified low-density lipoproteins (LDL) vigorously via scavenger receptor A (SR-A) to become foam cells. In the present study, we found that Sac1, a member of the Sac family of phosphoinositide phosphatases, increases the protein level of SR-A and upregulates foam cell formation. Mouse macrophages (RAW264.7) were transfected with short hairpin RNAs (shRNAs) against Sac1. Sac1 knockdown decreased cell surface SR-A levels and impaired acetylated LDL-induced foam cell formation. Transfection of Sac1-knockdown cells with shRNA-resistant flag-Sac1 effectively rescued the expression of SR-A. Glycosylation of SR-A was largely attenuated by Sac1 knockdown, but neither mRNA expression nor protein degradation of SR-A were affected. These results suggest that Sac1 maintains SR-A protein levels by modulating SR-A glycosylation.

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) specifically catalyzes the transfer of arachidonoyl-CoA to lysophosphoinositides. LPIAT

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling has been implicated in the anti-inflammatory response in a mouse model of endotoxemia and sepsis. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a), which dephosphorylates PtdIns(3,4)P2 to PtdIns(3)P, in bacterial infections. We prepared myeloid cell-specific Inpp4a-conditional knockout mice. Macrophages from these mice showed increased Akt phosphorylation and reduced production of inflammatory cytokines in response to LPS or Escherichia coli in vitro. The Inpp4a knockout mice survived for a shorter time than wild type mice after i.p. infection with E. coli, with less production of inflammatory cytokines. Additionally, E. coli clearance from blood and lung was significantly impaired in the knockout mice. A likely mechanism is that the Inpp4a-catalyzed dephosphorylation of PtdIns(3,4)P2 down-regulates Akt pathways, which, in turn, increases the production of inflammatory mediators. This mechanism at least fits the decreased E. coli clearance and short survival in the Inpp4a knockout mice.

TMEM55a (also known as PIP4P2) is an enzyme that dephosphorylates the phosphatidylinositol (PtdIns) PtdIns(4,5)P2 to form PtdIns(5)P in vitro However, the in vivo conversion of the polyphosphoinositide into PtdIns(5)P by the phosphatase has not yet been demonstrated, and the role of TMEM55a remains poorly understood. Here, we found that mouse macrophages (Raw264.7) deficient in TMEM55a showed an increased engulfment of large particles without affecting the phagocytosis of Escherichia coli Transfection of a bacterial phosphatase with similar substrate specificity to TMEM55a, namely IpgD, into Raw264.7 cells inhibited the engulfment of IgG-erythrocytes in a manner dependent on its phosphatase activity. In contrast, cells transfected with PIP4K2a, which catalyzes PtdIns(4,5)P2 production from PtdIns(5)P, increased phagocytosis. Fluorescent TMEM55a transfected into Raw264.7 cells was found to mostly localize to the phagosome. The accumulation of PtdIns(4,5)P2, PtdIns(3,4,5)P3 and F-actin on the phagocytic cup was increased in TMEM55a-deficient cells, as monitored by live-cell imaging. Phagosomal PtdIns(5)P was decreased in the knockdown cells, but the augmentation of phagocytosis in these cells was unaffected by the exogenous addition of PtdIns(5)P. Taken together, these results suggest that TMEM55a negatively regulates the phagocytosis of large particles by reducing phagosomal PtdIns(4,5)P2 accumulation during cup formation.

The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in Raw 264.7 macrophages. The clearance of PIs was much faster when the phagocytosed particles were coated with IgG. The effect of IgG was not observed in the macrophages deficient in FcγRIIb, an inhibitory IgG receptor. To identify the lipid phosphatases responsible for the FcγRIIb-accelerated PI clearance, we prepared a panel of lipid phosphatase-deficient cells. The lack of a PI 5-phosphatase Src homology 2 domain-containing inositol-5-phosphatase (SHIP)1 or SHIP2 impaired the FcγRIIb-accelerated clearance of PIs. The lack of a PI 4-phosphatase Inpp4a also impaired the accelerated PIs clearance. In the FcγRIIb- and Inpp4a-deficient cells, acidification of the formed phagosome was slowed. These results suggested that FcγRIIb drives the sequential dephosphorylation system comprising SHIPs and Inpp4a, and accelerates phagosome acidification.

The findings of this study suggest that the phosphoinositide phosphatase Sac3 maintains the protein level of scavenger receptor A (SR-A) and regulates foam cell formation. RAW264.7 macrophages were transfected with short hairpin RNAs that target Sac3. The knockdown decreased the level of the cell surface SR-A and suppressed the acetylated low density lipoprotein-induced foam cell formation. The associated regulator of PIKfyve (ArPIKfyve) is a scaffold protein that protects Sac3 from proteasome-dependent degradation. The knockdown of ArPIKfyve decreased Sac3, cell surface SR-A, and foam cell formation. The knockdown of PIKfyve had no effect on SR-A protein levels. These results suggest that the ArPIKfyve-Sac3 complex regulates SR-A protein levels independently of its effect on PIKfyve activity.

Phosphoinositide 5′-phosphatases have been implicated in the regulation of phagocytosis. However, their precise roles in the phagocytic process are poorly understood. We prepared RAW264.7 macrophages deficient in Inpp5e (shInpp5e) to clarify the role of this lipid phosphatase. In the shInpp5e cells, the uptake of solid particles was increased and the rate of phagosome acidification was accelerated. As expected, levels of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were increased and decreased respectively, on the forming phagocytic cups of these cells. Unexpectedly, the most prominent consequence of the Inpp5e deficiency was the decreased accumulation of PtdIns3P and Rab5 on the phagosome. The expression of a constitutively active form of Rab5b in the shInpp5e cells rescued the PtdIns3P accumulation. Rab20 has been reported to regulate the activity of Rabex5, a guanine nucleotide exchange factor for Rab5. The association of Rab20 with the phagosome was remarkably abrogated in the shInpp5e cells. Over-expression of Rab20 increased phagosomal PtdIns3P accumulation and delayed its elimination. These results suggest that Inpp5e, through functional interactions with Rab20 on the phagosome, activates Rab5, which, in turn, increases PtdIns3P and delays phagosome acidification.

Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P.