Phosphatidylinositol phospholipase C mediates carbon sensing and vegetative nuclear duplication rates in Aspergillus nidulans

In this work, we disrupted one of three putative phosphatidylinositol phospholipase C genes of Aspergillus nidulans and studied its effect on carbon source sensing linked to vegetative mitotic nuclear division. We showed that glucose does not affect nuclear division rates during early vegetative conidial germination (6–7 h) in either the wild type or the plcA-deficient mutant. Only after 8 h of cultivation on glucose did the mutant strain present some decrease in nuclear duplication. However, decreased nuclear division rates were observed in the wild type when cultivated in media amended with polypectate, whereas our plcA-deficient mutant did not show slow nuclear duplication rates when grown on this carbon source, even though it requires induction and secretion of multiple pectinolytic enzymes to be metabolized. Thus, plcA appears to be directly linked to high-molecular-weight carbon source sensing.

The glycosylphosphatidylinositol anchor is a major determinant of prion binding and replication

The prion diseases occur following the conversion of the cellular prion protein (PrPC) into an alternatively folded, disease-associated isoform (PrPSc). However, the spread of PrPSc from cell to cell is poorly understood. In the present manuscript we report that soluble PrPSc bound to and replicated within both GT1 neuronal cells and primary cortical neurons. The capacity of PrPSc to bind and replicate within cells was significantly reduced by enzymatic modification of its GPI (glycosylphosphatidylinositol) anchor. Thus PrPSc that had been digested with phosphatidylinositol-phospholipase C bound poorly to GT1 cells or cortical neurons and did not result in PrPSc formation in recipient cells. PrPSc that had been digested with phospholipase A2 (PrPSc-G-lyso-PI) bound readily to GT1 cells and cortical neurons but replicated less efficiently than mock-treated PrPSc. Whereas the addition of PrPSc increased cellular cholesterol levels and was predominantly found within lipid raft micro-domains, PrPSc-G-lyso-PI did not alter cholesterol levels and most of it was found outside lipid rafts. We conclude that the nature of the GPI anchor attached to PrPSc affected the binding of PrPSc to neurons, its localization to lipid rafts and its ability to convert endogenous PrPC.

Hypoxia-induced mitogenic factor/FIZZ1 induces intracellular calcium release through the PLC-IP3 pathway

Hypoxia-induced mitogenic factor (HIMF), also known as “found in inflammatory zone 1” (FIZZ1) or resistin-like molecule-α (RELMα), is a profound vasoconstrictor of the pulmonary circulation and a strong mitogenic factor in pulmonary vascular smooth muscle. To further understand the mechanism of these contractile and mitogenic responses, we examined the effect of HIMF on intracellular Ca2+ in human pulmonary artery smooth muscle cells (SMC). Ca2+ imaging in fluo 4-loaded human pulmonary artery SMC revealed that recombinant murine HIMF increased intracellular Ca2+ concentration ([Ca2+]i) in a sustained and oscillatory manner. This increase occurred independent of extracellular Ca2+ influx. Pretreatment of human pulmonary artery SMC with U-73122, a specific inhibitor of phosphatidylinositol-phospholipase C (PLC) completely prevented the HIMF-induced Ca2+ signal. The [Ca2+]i increase was also abolished by pretreatment with 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP3) receptor antagonist. Ryanodine pretreatment did not affect initiation of [Ca2+]i activation or internal release but reduced [Ca2+]i at the plateau phase. Pretreatment with the Gαi-specific inhibitor pertussis toxin and the Gαs-specific inhibitor NF-449 did not block the Ca2+ signal. Knockdown of Gαq/11 expression did not prevent Ca2+ release, but the pattern of Ca2+ release changed from the sustained oscillatory transients with prolonged plateau to a series of short [Ca2+]i transients that return to baseline. However, pretreatment with the tyrosine kinase inhibitor genistein completely inhibited the internal Ca2+ release. These results demonstrate that HIMF can stimulate intracellular Ca2+ release in human pulmonary artery SMC through the PLC signaling pathway in an IP3- and tyrosine phosphorylation-dependent manner and that Gαq/11 protein-coupled receptor and ryanodine receptor contribute to the increase of [Ca2+]i.

Requirement of Glycosylphosphatidylinositol Anchor of Cripto-1 for trans Activity as a Nodal Co-receptor

Cripto-1 (CR-1) has an indispensable role as a Nodal co-receptor for patterning of body axis in embryonic development. CR-1 is reported to have a paracrine activity as a Nodal co-receptor, although CR-1 is primarily produced as a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Regulation of cis and trans function of CR-1 should be important to establish the precise body patterning. However, the mechanism by which GPI-anchored CR-1 can act in trans is not well known. Here we confirmed the paracrine activity of CR-1 by fluorescent cell-labeling and immunofluorescent staining. We generated COOH-terminal-truncated soluble forms of CR-1 based on the attachment site for the GPI moiety (ω-site), which we identified in the present study. GPI-anchored CR-1 has a significantly higher activity than COOH-terminal-truncated soluble forms to induce Nodal signal in trans as well as in cis. Moreover, transmembrane forms of CR-1 partially retained their ability to induce Nodal signaling only when type I receptor Activin-like kinase 4 was overexpressed. NTERA2/D1 cells, which express endogenous CR-1, lost the cell-surface expression of CR-1 after phosphatidylinositol-phospholipase C treatment and became refractory to stimulation of Nodal. These observations suggest that GPI attachment of CR-1 is required for the paracrine activity as a Nodal co-receptor.

GPI-linked endothelial CD14 contributes to the detection of LPS

The inflammatory endothelial response to LPS is critical to the host's surviving a gram-negative bacterial infection. In this study we investigated whether human endothelial cells express the functional coreceptor for LPS, CD14, and most importantly whether it is glycosylphosphatidylinositol (GPI) linked. We also examined whether plasma proteins could reconstitute an LPS response in CD14-inhibited endothelium. RT-PCR- and CD14-specific MAbs demonstrated CD14 expression on primary human umbilical vein endothelial cells (HUVEC) but not passaged HUVEC. The amino acid sequence of endothelial CD14 was 99% homologous to CD14 on monocytes. Endothelium responded to relatively low levels of LPS in the absence of plasma, and this was entirely dependent on CD14. Removal of GPI-linked proteins with phosphatidylinositol-phospholipase C prevented LPS detection and subsequent protein synthesis (E-selectin expression). Endothelial CD14 was sufficient to initiate functional leukocyte recruitment, an event inhibited by blocking its LPS binding epitope and also by removing CD14 from the endothelial surface. Plasma proteins restored only ∼30% of the LPS response in CD14-inhibited endothelium. In conclusion, our results strongly support an important role for endothelial membrane CD14 in the activation of endothelium for leukocyte recruitment.