Hisako Hikiji

The roles of prostanoids, leukotrienes, and platelet-activating factor in bone metabolism and disease

The production of a variety of lipid mediators is enhanced in bone-resorptive diseases such as osteoporosis, rheumatoid arthritis, osteoarthritis, and periodontitis. Prostaglandin E(2) (PGE(2)) is one of the most notable lipid mediators of bone remodeling, and has been linked clinically to many bone-resorptive diseases. In vitro studies with bone cell cultures have demonstrated that the bone-resorptive activity of PGE(2), which is mediated by receptor activator of NF-kappaB ligand (RANKL), is key for the induction of osteoclast formation. Furthermore, interleukin (IL)-1- and IL-6-stimulated bone resorption involves PGE(2) production. In addition to its bone-resorptive effects, PGE(2) promotes bone formation in vitro by stimulating osteoblastic proliferation and differentiation. The multifaceted nature of PGE(2) makes it difficult to discern its role during bone remodeling. Leukotrienes (LTs), and particularly LTB(4), have also been implicated in bone remodeling and disease-specifically in rheumatoid arthritis. Moreover, recent studies from our laboratory have shown that platelet-activating factor (PAF) receptor-deficient mice develop only mild osteoporosis. Osteoclast survival in these mice is shortened and osteoclastic bone resorption is impaired. This review article focuses on these families of lipids and their function during bone metabolism and disease.

Direct action of nitric oxide on osteoblastic differentiation

The effect of nitric oxide (NO) on osteoblastic differentiation was examined in cultured mouse osteoblasts. Interleukin‐1β and tumor necrosis factor‐α expressed inducible NO synthase gene with little effect on constitutive NO synthase gene. These cytokines increased NO production, which was inhibited by l‐NMMA pretreatment, and decreased alkaline phosphatase (AIPase) activity, which was not restored by l‐NMMA. Furthermore, NO donors, sodium nitroprusside and NONOate dose‐dependently elevated AIPase activity and expression of osteocalcin gene. These results suggest that NO directly facilitates osteoblastic differentiation and the cytokine‐induced inhibition of AIPase activity is mediated via mechanism other than NO.

A distinctive role of the leukotriene B4 receptor BLT1 in osteoclastic activity during bone loss

Although leukotriene B4 (LTB4) is produced in various inflammatory diseases, its functions in bone metabolism remain unknown. Using mice deficient in the high-affinity LTB4 receptor BLT1, we evaluated the roles of BLT1 in the development of two bone resorption models, namely bone loss induced by ovariectomy and lipopolysaccharide. Through observations of bone mineral contents and bone morphometric parameters, we found that bone resorption in both models was significantly attenuated in BLT1-deficient mice. Furthermore, osteoclasts from BLT1-deficient mice showed reduced calcium resorption activities compared with wild-type osteoclasts. Osteoclasts expressed BLT1, but not the low-affinity LTB4 receptor BLT2, and produced LTB4. LTB4 changed the cell morphology of osteoclasts through the BLT1-Gi protein-Rac1 signaling pathway. Given the causal relationship between osteoclast morphology and osteoclastic activity, these findings suggest that autocrine/paracrine LTB4 increases the osteoclastic activity through the BLT1-Gi protein-Rac1 signaling pathway. Inhibition of BLT1 functions may represent a strategy for preventing bone resorption diseases.

Absence of platelet-activating factor receptor protects mice from osteoporosis following ovariectomy

While platelet-activating factor (PAF) is produced in various diseases associated with bone resorption, its functions in bone metabolism remain unknown. Using PAF receptor-deficient mice, we evaluated the role of PAF in the development of bone resorption following ovariectomy, a model of postmenopausal osteoporosis. Through observations of bone mineral density and histomorphometric parameters, it was found that bone resorption was markedly attenuated in PAF receptor-deficient mice, indicating that PAF links estrogen depletion and osteoporosis in vivo. Osteoclasts expressed higher amounts of the enzymes required for PAF biosynthesis than osteoblasts. TNF-alpha and IL-1beta increased the acetyl-coenzyme A:lyso-PAF acetyltransferase activity in osteoclasts. Osteoclasts, but not osteoblasts, expressed the functional PAF receptor. PAF receptor stimulation prolonged the survival of osteoclasts in vitro. Furthermore, osteoclasts treated with a PAF receptor antagonist, and also those from PAF receptor-deficient mice, showed reductions in survival rate and Ca resorption activity. Consistently, in organ cultures, bone resorption was significantly suppressed by a PAF receptor antagonist treatment or genetic PAF receptor deficiency. Thus, these results suggest that, through the inflammatory cytokines, estrogen depletion enhances PAF production as a unique autocrine factor for osteoclast functions. Inhibition of PAF function might pave the way for a new strategy to prevent postmenopausal bone loss without disturbing osteoblast functions.

P130Cas, Crk-associated substrate, plays important roles in osteoclastic bone Resorption

p130Cas, Crk‐associated substrate (Cas), is an adaptor/scaffold protein that plays a central role in actin cytoskeletal reorganization. We previously reported that p130Cas is not tyrosine‐phosphorylated in osteoclasts derived from Src‐deficient mice, which are congenitally osteopetrotic, suggesting that p130Cas serves as a downstream molecule of c‐Src and is involved in osteoclastic bone resorption. However, the physiological role of p130Cas in osteoclasts has not yet been confirmed because the p130Cas‐deficient mice displayed embryonic lethality. Osteoclast‐specific p130Cas conditional knockout (p130CasΔOCL–) mice exhibit a high bone mass phenotype caused by defect in multinucleation and cytoskeleton organization causing bone resorption deficiency. Bone marrow cells from p130CasΔOCL– mice were able to differentiate into osteoclasts and wild‐type cells in vitro. However, osteoclasts from p130CasΔOCL– mice failed to form actin rings and resorb pits on dentine slices. Although the initial events of osteoclast attachment, such as β3‐integrin or Src phosphorylation, were intact, the Rac1 activity that organizes the actin cytoskeleton was reduced, and its distribution was disrupted in p130CasΔOCL– osteoclasts. Dedicator of cytokinesis 5 (Dock5), a Rho family guanine nucleotide exchanger, failed to associate with Src or Pyk2 in osteoclasts in the absence of p130Cas. These results strongly indicate that p130Cas plays pivotal roles in osteoclastic bone resorption.

Molecular and pharmacological characteristics of transient voltage-dependent K+ currents in cultured human pulmonary arterial smooth muscle cells

The A‐type voltage‐dependent K+ current (IA) has been identified in several types of smooth muscle cells including the pulmonary artery (PA), but little is known about the pharmacological and molecular characteristics of IA in human pulmonary arterial smooth muscle cells (hPASMCs). We investigated IA expressed in cultured PASMCs isolated from the human main pulmonary artery, using patch‐clamp techniques, reverse transcriptase–polymerase chain reaction (RT–PCR), quantitative real‐time RT–PCR and immunocytochemical studies. With high EGTA and ATP in the pipette, the outward currents were dominated by a transient K+ current (IA), followed by a relatively small sustained outward current (IK). IA was inhibited by 4‐aminopyridine (4‐AP) concentration‐dependently, and could be separated pharmacologically into two components by tetraethylammonium (TEA) sensitivity. A component was sensitive to TEA, and the second component was insensitive to TEA. IA was inhibited by blood depressing substrate (BDS)‐II, a specific blocker of KV3.4 subunit, and phrixotoxin‐II, a specific blocker of KV4.2 and 4.3. Flecainide inhibited IA concentration‐dependently, but it inhibited it preferentially in the presence of TEA (TEA‐insensitive IA). Systematic screening of expression of KV genes using RT–PCR showed the definite presence of transcripts of the IA‐encoding genes for KV3.4, KV4.1, KV4.2 and KV4.3 as well as the IK‐encoding genes for KV1.1, KV1.5 and KV2.1. The real‐time RT–PCR analysis showed that the relative abundance of the encoding genes of IA α‐subunit and KV channel‐interacting proteins (KChIPs) was KV4.2>KV3.4>KV4.3 (long)>KV4.1, and KChIP3≫KChIP2, respectively. The presence of KV3.4, KV4.2 and KV4.3 proteins was also demonstrated by immunocytochemical studies, and confirmed by immunohistochemical staining using intact human PA sections. These results suggest that IA in cultured hPASMCs consists of two kinetically and pharmacologically distinct components, probably KV3.4 and KV4 channels.

Essential Role of Lysophosphatidylcholine Acyltransferase 3 in the Induction of Macrophage Polarization in PMA‐Treated U937 Cells

Lysophospholipid acyltransferases (LPLATs) regulate the diversification of fatty acid composition in biological membranes. Lysophosphatidylcholine acyltransferases (LPCATs) are members of the LPLATs that play a role in inflammatory responses. M1 macrophages differentiate in response to lipopolysaccharide (LPS) and are pro‐inflammatory, whereas M2 macrophages, which differentiate in response to interleukin‐4 (IL‐4), are anti‐inflammatory and involved in homeostasis and wound healing. In the present study, we showed that LPCATs play an important role in M1/M2‐macrophage polarization. LPS changed the shape of PMA‐treated U937 cells from rounded to spindle shaped and upregulated the mRNA and protein expression of the M1 macrophage markers CXCL10, TNF‐α, and IL‐1β. IL‐4 had no effect on the shape of PMA‐treated U937 cells and upregulated the M2 macrophage markers CD206, IL‐1ra, and TGF‐β in PMA‐treated U937 cells. These results suggest that LPS and IL‐4 promote the differentiation of PMA‐treated U937 cells into M1‐ and M2‐polarized macrophages, respectively. LPS significantly downregulated the mRNA expression of LPCAT3, one of four LPCAT isoforms, and suppressed its enzymatic activity toward linoleoyl‐CoA and arachidonoyl‐CoA in PMA‐treated U937 cells. LPCAT3 knockdown induced a spindle‐shaped morphology typical of M1‐polarized macrophages, and increased the secretion of CXCL10 and decreased the levels of CD206 in IL‐4‐activated U937 cells. This indicates that knockdown of LPCAT3 shifts the differentiation of PMA‐treated U937 cells to M1‐polarized macrophages. Our findings suggest that LPCAT3 plays an important role in M1/M2‐macrophage polarization, providing novel potential therapeutic targets for the regulation of immune and inflammatory disorders. J. Cell. Biochem. 116: 2840–2848, 2015.

Correction of cleft lip nasal deformity in Orientals with a cantilevered iliac bone graft.

We describe our technique for correcting a nasal deformity associated with cleft lip in oriental people. Cantilevered iliac bone grafts are used to provide additional structural support and to achieve the desired nasal projection and profile. Augmentation of the nasal bridge creates the illusion of a narrower nose. This technique was used in 20 patients with severe nasal deformities. Clinically and radiographically it consistently produced good, long-lasting results.We describe our technique for correcting a nasal deformity associated with cleft lip in oriental people. Cantilevered iliac bone grafts are used to provide additional structural support and to achieve the desired nasal projection and profile. Augmentation of the nasal bridge creates the illusion of a narrower nose. This technique was used in 20 patients with severe nasal deformities. Clinically and radiographically it consistently produced good, long-lasting results.

Evaluation and analysis of formation of bone at the palate in patients with cleft lip and palate after palatoplasty based on computed tomograms and three-dimensional data

Abstract There are various techniques for palatoplasty, but no studies of postoperative osteogenesis at the palatal fissure. In the cranial and maxillofacial region it is thought to develop from the periosteum, so palatoplasty with mucoperiosteal flaps may encourage new bone to form at the fissure. We evaluated the status of osteogenesis in the hard palate after palatoplasty on computed tomograms (CT). We studied 29 patients (22 boys and 7 girls) with unilateral cleft lip and palate who had pushback palatoplasty with the use of CT obtained between May 2003 and March 2007. Age at the time of operation was recorded. The width of the palatal fissure at the first premolar, the first molar, and the maxillary posterior region were measured on coronal CT. The mean (SD) age at the time of palatoplasty was 16 (2) months. The mean (SD) width of the fissure at the first molar was 3.96 (3.1) mm, and bony union was seen in four patients. The width of the fissure was significantly less at the first molar than at the other sites (p = 0.006). The shape of the margin of the fissure was irregular in nearly all patients. The width of the fissure at the first molar became significantly less, suggesting that osteogenesis had occurred. In some patients the height of the fissure differed. Given the results of previous studies, bony regeneration from the periosteum most likely happens together with regeneration from the margins of the fissure.

Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell–derived neurons

Glycerophospholipids, which are components of biomembranes, are formed de novo by the Kennedy pathway and subsequently mature through the Lands cycle. Lysophospholipid acyltransferases (LPLATs) are key enzymes in both pathways and influence the fatty acid composition of biomembranes. Neuronal differentiation is characterized by neurite outgrowth, which requires biomembrane biosynthesis. However, the role of LPLATs in neuronal differentiation remains unknown. In this study, we examined whether LPLATs are involved in neuronal differentiation using all‐trans‐retinoic acid (ATRA)‐treated P19C6 cells. In these cells, mRNA levels of lysophosphatidylethanolamine acyltransferase (LPEAT)‐1/membrane‐bound O‐acyltransferase (MBOAT)‐1 were higher than those in undifferentiated cells. LPEAT enzymatic activity increased with 16:0‐ and 18:1‐CoA as acyl donors. When LPEAT1/MBOAT1 was knocked down with small interfering RNA (siRNA), outgrowth of neurites and expression of neuronal markers decreased in ATRA‐treated P19C6 cells. Voltage‐dependent calcium channel activity was also suppressed in these cells transfected with LPEAT1/MBOAT1 siRNA. These results suggest that LPEAT1/MBOAT1 plays an important role in neurite outgrowth and function.—Tabe, S., Hikiji, H., Ariyoshi, W., Hashidate‐Yoshida, T., Shindou, H., Okinaga, T., Shimizu, T., Tominaga, K., Nishihara, T. Lysophosphatidylethanolamine acyltransferase 1/membrane‐bound O‐acyltransferase 1 regulates morphology and function of P19C6 cell‐derived neurons. FASEB J. 30, 2591‐2601 (2016). www.fasebj.org