Toshifumi Sugatani

The Mechanism of Phosphorus as a Cardiovascular Risk Factor in CKD

Hyperphosphatemia and vascular calcification have emerged as cardiovascular risk factors among those with chronic kidney disease. This study examined the mechanism by which phosphorous stimulates vascular calcification, as well as how controlling hyperphosphatemia affects established calcification. In primary cultures of vascular smooth muscle cells derived from atherosclerotic human aortas, activation of osteoblastic events, including increased expression of bone morphogenetic protein 2 (BMP-2) and the transcription factor RUNX2, which normally play roles in skeletal morphogenesis, was observed. These changes, however, did not lead to matrix mineralization until the phosphorus concentration of the media was increased; phosphorus stimulated expression of osterix, a second critical osteoblast transcription factor. Knockdown of osterix with small interference RNA (siRNA) or antagonism of BMP-2 with noggin prevented matrix mineralization in vitro. Similarly, vascular BMP-2 and RUNX2 were upregulated in atherosclerotic mice, but significant mineralization occurred only after the induction of renal dysfunction, which led to hyperphosphatemia and increased aortic expression of osterix. Administration of oral phosphate binders or intraperitoneal BMP-7 decreased expression of osterix and aortic mineralization. It is concluded that, in chronic kidney disease, hyperphosphatemia stimulates an osteoblastic transcriptional program in the vasculature, which is mediated by osterix activation in cells of the vascular tunica media and neointima.

Impaired Micro-RNA Pathways Diminish Osteoclast Differentiation and Function

Micro-RNAs (miRNAs) are important in regulating cell fate determination because many of their target mRNA transcripts are engaged in cell proliferation, differentiation, and apoptosis. DGCR8, Dicer, and Ago2 are essential factors for miRNA homeostasis. Here we show that these three factors have critical roles in osteoclast differentiation and function. Gene silencing of DGCR8, Dicer, or Ago2 by small interfering RNA revealed global inhibition of osteoclast transcription factor expression and function, decreased osteoclastogenesis, and decreased bone resorption in vitro. In vivo, CD11b+-cre/Dicer-null mice had mild osteopetrosis caused by decreased osteoclast number and bone resorption. These results suggest that miRNAs play important roles in differentiation and function of osteoclasts in vitro and in vivo. We found a novel mechanism mediating these results in which PU.1, miRNA-223, NFI-A, and the macrophage colony-stimulating factor receptor (M-CSFR) are closely linked through a positive feedback loop. PU.1 stimulates miRNA-223 expression, and this up-regulation is implicated in stimulating differentiation and function of osteoclasts through negative regulation of NFI-A levels. Down-regulation of NFI-A levels is important for expression of the M-CSFR, which is critical for osteoclast differentiation and function. NFI-A overexpression decreased osteoclast formation and function with down-regulation of M-CSFR levels. Forced expression of the M-CSFR in M-CSF-dependent bone marrow macrophages from Dicer-deficient mice rescued osteoclast differentiation with up-regulation of PU.1 levels. Our studies provide new molecular mechanisms controlling osteoclast differentiation and function by the miRNA system and specifically by miRNA-223, which regulates NFI-A and the M-CSFR levels.

MicroRNA-223 is a key factor in osteoclast differentiation

MicroRNAs (miRNAs) are a class of noncording RNAs that control gene expression by translational inhibition and messenger RNAs (mRNAs) degradation in plants and animals. Although miRNAs have been implicated in developmental and homeostatic events of vertebrates and invertebrates, the role of miRNAs in bone metabolism has not been explored. Here, we show that microRNA‐223 (miR‐223) is expressed in RAW264.7 cells, mouse osteoclast precursor cell lines, and plays a critical role in osteoclast differentiation. We constructed miR‐223 short interfering RNA (siRNA) or precursor miR‐223 (pre‐miR‐223) overexpression retroviral vectors, and established miR‐223 knockdown by siRNA or pre‐miR‐223 overexpression in stably infected RAW264.7 cells. Tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells were observed in miR‐223 knockdown cells as well as control cells. In contrast, pre‐miR‐223 overexpression completely blocked TRAP‐positive multinucleated cell formation compared with control cells. Apoptotic cells were not observed in this study. Our results indicate that miR‐223 plays an essential role during osteoclast differentiation, and miR‐223 might be a viable therapeutic target for a range of bone metabolic disorders with excess osteoclast activity. J. Cell. Biochem. 101: 996–999, 2007.

A microRNA expression signature of osteoclastogenesis

MicroRNAs (miRs) are small noncoding RNAs that principally function in the spatiotemporal regulation of protein translation in animal cells. Although emerging evidence suggests that some miRs play important roles in osteoblastogenesis and skeletal homeostasis, much less is known in osteoclastogenesis. Here, we show that receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis is mediated by miR-21. MiR-21 was identified as an miR expression signature of RANKL-induced osteoclastogenesis that down-regulates programmed cell death 4 (PDCD4) protein levels. Diminished PDCD4 removes a repression from c-Fos, a critical transcription factor for osteoclastogenesis and osteoclast-specific downstream target genes. In addition, RANKL-induced c-Fos up-regulates miR-21 gene expression. Bone marrow-derived monocyte/macrophage precursors deficient of DiGeorge syndrome critical region gene 8, an RNA binding protein associated with miR biogenesis, and Dicer, an endoribonuclease in the RNaseIII family associated with miR biogenesis, possessed significantly decreased miR-21 levels and increased PDCD4 protein levels so that RANKL-induced osteoclastogenesis was impaired in those cells. However, forced expression of miR-21 rescued osteoclast development because of down-regulation of PDCD4 protein expression levels. Thus, our studies provide a new molecular mechanism, including a positive feedback loop of c-Fos/miR-21/PDCD4, regulating osteoclastogenesis.

Early chronic kidney disease-mineral bone disorder stimulates vascular calcification

The chronic kidney disease-mineral and bone disorder (CKD-MBD) syndrome is an extremely important complication of kidney diseases. Here we tested whether CKD-MBD causes vascular calcification in early kidney failure by developing a mouse model of early CKD in a background of atherosclerosis stimulated arterial calcification. CKD equivalent in glomerular filtration reduction to human CKD stage 2 stimulated early vascular calcification and inhibited the tissue expression of α-klotho (klotho) in the aorta. In addition, osteoblast transition in the aorta was stimulated by early CKD as shown by the expression of the critical transcription factor, RUNX2. The ligand associated with the klotho-fibroblast growth factor receptor complex, FGF23, was found to be expressed in the vascular media of sham operated mice. Its expression was decreased in early CKD. Increased circulating levels of the osteocyte secreted proteins, FGF23, and sclerostin may have been related to increased circulating klotho levels. Finally, we observed low turnover bone disease with a reduction in bone formation rates more than bone resorption. Thus, the CKD-MBD, characterized by cardiovascular risk factors, vascular calcification, increased circulating klotho, FGF23 and sclerostin levels, and low turnover renal osteodystrophy, was established in early CKD. Early CKD caused a reduction of vascular klotho, stimulated vascular osteoblastic transition, increased osteocytic secreted proteins, and inhibited skeletal modeling producing the CKD-MBD.

CKD-Induced Wingless/Integration1 Inhibitors and Phosphorus Cause the CKD–Mineral and Bone Disorder

In chronic kidney disease, vascular calcification, renal osteodystrophy, and phosphate contribute substantially to cardiovascular risk and are components of CKD-mineral and bone disorder (CKD-MBD). The cause of this syndrome is unknown. Additionally, no therapy addresses cardiovascular risk in CKD. In its inception, CKD-MBD is characterized by osteodystrophy, vascular calcification, and stimulation of osteocyte secretion. We tested the hypothesis that increased production of circulating factors by diseased kidneys causes the CKD-MBD in diabetic mice subjected to renal injury to induce stage 2 CKD (CKD-2 mice). Compared with non-CKD diabetic controls, CKD-2 mice showed increased renal production of Wnt inhibitor family members and higher levels of circulating Dickkopf-1 (Dkk1), sclerostin, and secreted klotho. Neutralization of Dkk1 in CKD-2 mice by administration of a monoclonal antibody after renal injury stimulated bone formation rates, corrected the osteodystrophy, and prevented CKD-stimulated vascular calcification. Mechanistically, neutralization of Dkk1 suppressed aortic expression of the osteoblastic transcription factor Runx2, increased expression of vascular smooth muscle protein 22-α, and restored aortic expression of klotho. Neutralization of Dkk1 did not affect the elevated plasma levels of osteocytic fibroblast growth factor 23 but decreased the elevated levels of sclerostin. Phosphate binder therapy restored plasma fibroblast growth factor 23 levels but had no effect on vascular calcification or osteodystrophy. The combination of the Dkk1 antibody and phosphate binder therapy completely treated the CKD-MBD. These results show that circulating Wnt inhibitors are involved in the pathogenesis of CKD-MBD and that the combination of Dkk1 neutralization and phosphate binding may have therapeutic potential for this disorder.

Activin A stimulates IκB-α/NFκB and rank expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor‐κB (NFκB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IκB‐α, and stimulates nuclear translocation of NFκB and receptor activator of nuclear factor‐κB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IκB‐α and induced nuclear translocation of phosphorylated‐NFκB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL‐ and M‐CSF‐stimulated nuclear translocation of NFκB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M‐CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFκB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis. J. Cell. Biochem. 90: 59–67, 2003.

PTEN Regulates RANKL- and Osteopontin-stimulated Signal Transduction during Osteoclast Differentiation and Cell Motility

PTEN (also known as MMAC-1 or TEP-1) is a frequently mutated tumor suppressor gene in human cancer. PTEN functions have been identified in the regulation of cell survival, growth, adhesion, migration, and invasiveness. Here, we characterize the diverse signaling networks modulated by PTEN in osteoclast precursors stimulated by RANKL and osteopontin (OPN). RANKL dose-dependently stimulated transient activation of Akt before activation of PTEN, consistent with a role for PTEN in decreasing Akt activity. PTEN overexpression blocked RANKL-activated Akt stimulated survival and osteopontin-stimulated cell migration while a dominant-negative PTEN increased the actions of RANKL and OPN. PTEN overexpression suppressed RANKL-mediated osteoclast differentiation and OPN-stimulated cell migration. The PTEN dominant-negative constitutively induced osteoclast differentiation and cell migration. Our data demonstrate multiple roles for PTEN in RANKL-induced osteoclast differentiation and OPN-stimulated cell migration in RAW 264.7 osteoclast precursors.

Down‐regulation of miR‐21 biogenesis by estrogen action contributes to osteoclastic apoptosis

Estrogen inhibits osteoclastogenesis and induces osteoclastic apoptosis; however, the molecular mechanisms remain controversial. Recently, a group has demonstrated that osteoclasts are a direct target for estrogen because estrogen stimulates transcription of the Fas Ligand (FasL) gene in osteoclasts, which in turn causes cell death through an autocrine mechanism. In contrast, other groups have shown that the cells are an indirect target for estrogen because estrogen fails to stimulate the transcription of that in osteoclasts. Thus, two quite different molecular mechanisms have been suggested to explain the effects of estrogen in osteoclastic apoptosis. Here we show that the proapoptotic effect of estrogen during osteoclastogenesis is regulated by a posttranscriptional increase in FasL production by down‐regulated microRNA‐21 (miR‐21) biogenesis. Previously, we reported that miR‐21 is highly expressed in osteoclastogenesis. We found that estrogen down‐regulates miR‐21 biogenesis so that FasL, the targets of miR‐21, protein levels are posttranscriptionally increased that induce osteoclastic apoptosis. Moreover, the gain‐of‐function of miR‐21 rescued the apoptosis. In addition, we failed to detect estrogen‐enhanced FasL levels at mRNA levels. Thus, osteoclastic survival is controlled by autocrine actions of FasL regulated by estrogen and miR‐21 plays a central role during estrogen‐controlled osteoclastogenesis. J. Cell. Biochem. 114: 1217–1222, 2013.

Myofibroma of the mandible: Clinicopathologic study and review of the literature

A case of mandibular myofibroma in a 2-month-old boy is presented. Including this case, 24 pediatric and 11 adult patients with maxillofacial myofibroma have been reported since 1981. Of the 24 pediatric patients, 15 (62.5%) had lesions affecting the mandible. The adult cases had no mandibular involvement. Histologic evaluation of the tissue specimen revealed an interlacing pattern of spindle-shaped cells with long oval nuclei. Tissue immunohistochemical staining found it to be reactive for antibodies directed against vimentin and alpha-smooth muscle actin, but not desmin, S-100 protein, neuron-specific enolase, or myoglobin. Electron microscopy examination revealed the following cells: myofibroblast-like cells, fibroblast-like cells, and intermediate cells that were similar to the fibroblast-like cells except for the presence of a few microfilaments. Myoblast-like cells were not seen.