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Phosphatidylinositol-4-phosphate 5-kinase type-1 gamma (Pip5k1c) is a lipid kinase that plays a pivotal role in the regulation of receptor-mediated calcium signaling in multiple tissues; however, its role in the skeleton is not clear. Here, we show that while deleting Pip5k1c expression in the mesenchymal stem cells using Prx1-Cre transgenic mice does not impair the intramembranous and endochondral ossification during skeletal development, it does cause osteopenia in adult mice, but not rapidly growing young mice. We found Pip5k1c loss dramatically decreases osteoblast formation and osteoid and mineral deposition, leading to reduced bone formation. Furthermore, Pip5k1c loss inhibits osteoblastic, but promotes adipogenic, differentiation of bone marrow stromal cells. Pip5k1c deficiency also impairs cytoplasmic calcium influx and inactivates the calcium/calmodulin-dependent protein kinase, which regulates levels of transcription factor Runx2 by modulating its stability and subsequent osteoblast and bone formation. In addition, Pip5k1c loss reduces levels of the receptor activator of nuclear factor-κB ligand, but not that of osteoprotegerin, its decoy receptor, in osteoblasts in bone and in sera. Finally, we found Pip5k1c loss impairs the ability of bone marrow stromal cells to support osteoclast formation of bone marrow monocytes and reduces the osteoclast precursor population in bone marrow, resulting in reduced osteoclast formation and bone resorption. We conclude Pip5k1c deficiency causes a low-turnover osteopenia in mice, with impairment of bone formation being greater than that of bone resorption. Collectively, we uncover a novel function and mechanism of Pip5k1c in the control of bone mass and identify a potential therapeutic target for osteoporosis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ceramide 1-phosphate (C1P) has been characterized as a sphingolipid that participates in cell signaling. Although C1P synthesis is thought to occur via phosphorylation of ceramide by ceramide kinase (CerK), the processes that regulate C1P formation and fate remain largely unknown. In this study we analyzed bone marrow-derived macrophages (BMDM) from CerK-null mice (Cerk(-/-)) and found significant levels of C1P, suggesting that previously unrecognized pathways may also lead to C1P formation. After these experiments we used an overexpression system, BMDM from Cerk(-/-) mice, and short-chain fluorescent ceramides to trace CerK-dependent formation of C1P. Because the ceramide analogs can also be converted to glucosylceramide (GlcCer) and sphingomyelin (SM), they allowed us to directly compare all three metabolites. We found that C1P produced by CerK is turned over rapidly when serum is removed or upon calcium chelation, whereas GlcCer and SM are stable under these conditions. We further demonstrated that ceramide must be transported to the Golgi complex to be phosphorylated by CerK. Inhibition of the ceramide transfer protein slowed down SM formation without decreasing C1P, suggesting an alternate route of ceramide transport. Other experiments indicated that, like GlcCer and SM, C1P traffics along the secretory pathway to reach the plasma membrane. Furthermore, in BMDM C1P was secreted more readily than was GlcCer or SM. Altogether, our results indicate that CerK is essential to C1P formation via phosphorylation of Cer, providing the first insights into mechanisms underlying ceramide access to CerK and C1P trafficking as well as clarifying C1P as a signaling entity.

Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.

Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1-/-tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1-/- mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1-/- mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.

Mevalonic aciduria (MA) and hyper-IgD and periodic fever syndrome (HIDS) are two inherited disorders both caused by depressed mevalonate kinase (MK) activity. MK is the first enzyme to follow the highly regulated 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR), which catalyzes the rate-limiting step in the isoprenoid/cholesterol biosynthesis pathway. In fibroblasts of MA patients, but not of HIDS patients, HMGR activity is elevated under normal growth conditions. This activity is down-regulated when cells are supplemented with the isoprenoid precursors geraniol, farnesol, and geranylgeraniol, and a mixture of 25-hydroxycholesterol and cholesterol. This indicates that the regulation of the pathway in these cells is not disturbed. The elevated HMGR activity is probably due to a shortage of non-sterol isoprenoid end products, as indicated by normal HMGR mRNA levels in MA fibroblasts. Furthermore, the HMGR activity in MA cells was more sensitive to geranylgeraniol suppression and less sensitive to sterol suppression than the HMGR activity in low density lipoprotein receptor-deficient cells. HMGR activity in MA cells was down-regulated also by addition of its product mevalonate to the culture medium. Thus, it appears that the elevation of mevalonate levels, which are high in MA patients and moderate in HIDS patients, allows the cells to compensate for the depressed MK activity. Indeed, the isoprenylation of Ras and RhoA protein appeared normal in HIDS and MA fibroblasts under normal conditions but showed increased sensitivity toward inhibition of HMGR by simvastatin. Our results indicate that MK-deficient cells maintain the flux through the isoprenoid/cholesterol biosynthesis pathway by elevating intracellular mevalonate levels.

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) is widespread in eukaryotic cells. In Saccharomyces cerevisiae, PtdIns(3,5)P(2) synthesis is catalyzed by the PtdIns3P 5-kinase Fab1p, and loss of this activity results in vacuolar morphological defects, indicating that PtdIns(3,5)P(2) is essential for vacuole homeostasis. We have therefore suggested that all Fab1p homologues may be PtdIns3P 5-kinases involved in membrane trafficking. It is unclear which phosphatidylinositol phosphate kinases (PIPkins) are responsible for PtdIns(3,5)P(2) synthesis in higher eukaryotes. To clarify how PtdIns(3,5)P(2) is synthesized in mammalian and other cells, we determined whether yeast and mammalian Fab1p homologues or mammalian Type I PIPkins (PtdIns4P 5-kinases) make PtdIns(3,5)P(2) in vivo. The recently cloned murine (p235) and Schizosaccharomyces pombe FAB1 homologues both restored basal PtdIns(3,5)P(2) synthesis in Deltafab1 cells and made PtdIns(3,5)P(2) in vitro. Only p235 corrected the growth and vacuolar defects of fab1 S. cerevisiae. A mammalian Type I PIPkin supported no PtdIns(3,5)P(2) synthesis. Thus, FAB1 and its homologues constitute a distinct class of Type III PIPkins dedicated to PtdIns(3,5)P(2) synthesis. The differential abilities of p235 and of SpFab1p to complement the phenotypic defects of Deltafab1 cells suggests that interaction(s) with other protein factors may be important for spatial and/or temporal regulation of PtdIns(3,5)P(2) synthesis. These results also suggest that p235 may regulate a step in membrane trafficking in mammalian cells that is analogous to its function in yeast.

Friedreich ataxia (FRDA) is associated with the expansion of a GAA. TTC triplet repeat in the first intron of the frataxin gene, resulting in reduced levels of frataxin mRNA and protein. To investigate the mechanisms by which the intronic expansion produces its effect, GAA.TTC repeats of various lengths (9 to 270 triplets) were cloned in both orientations in the intron of a reporter gene. Plasmids containing these repeats were transiently transfected into COS-7 cells. A length- and orientation-dependent inhibition of reporter gene expression was observed. RNase protection and Northern blot analyses showed very low levels of mature mRNA when longer GAA repeats were transcribed, with no accumulation of primary transcript. Replication of plasmids carrying long GAA.TTC tracts (approximately 250 triplets) was greatly inhibited in COS-7 cells compared with plasmids carrying (GAA.TTC)9 and (GAA.TTC)90. Replication inhibition was five times greater for the plasmid whose transcript contains (GAA)230 than for the plasmid whose transcript contains (UUC)270. Our in vivo investigation revealed that expanded GAA.TTC repeats from intron I of the FRDA gene inhibit transcription rather than post-transcriptional RNA processing and also interfere with replication. The molecular basis for these effects may be the formation of non-B DNA structures.

Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T). To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa. Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively. Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax. Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families. Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions. Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele. Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences. Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with stabilization of mevalonate binding.

We reported recently that mevalonate kinase (EC 2.7.1.36; ATP:mevalonate 5-phosphotransferase) that was isolated from rat liver and believed to be a cytosolic protein was localized in rat liver peroxisomes. In addition, we found that the mevalonate kinase monoclonal antibody used in the study also reacted with several other proteins present in the mitochondrial and cytosolic fractions. These findings raised the prospect of the presence of several isoenzymes of mevalonate kinase localized in different compartments of the cell. In the current study we produced four new polyclonal antibodies against different epitopes of mevalonate kinase to investigate the subcellular localization of the protein by several different approaches: (i) by analytical subcellular fractionation and immunoblotting of mevalonate kinase in the isolated subcellular fractions with the monospecific antibodies; (ii) by immunocryoelectron microscopy techniques; and (iii) by expressing the cDNA encoding mevalonate kinase in mammalian cells. The data obtained demonstrate that there is only one mevalonate kinase protein that is predominantly localized in peroxisomes. We also illustrate that the protein is targeted to and imported into peroxisomes. In addition, we show that in cells and tissues obtained from patients with peroxisomal deficiency diseases mevalonate kinase protein and its activity are severely reduced.