Lithium reduces orthodontically induced root resorption by suppressing cell death, hyalinization, and odontoclast formation in rats.

OBJECTIVES: To examine whether lithium suppresses orthodontically induced root resorption (OIRR) via two mechanisms (prevention of hyalinization in periodontal tissue and suppression of odontoclasts) and to investigate the changes in the periodontal tissue and alveolar bone, focusing on the appearance of cell death, hyalinization, and odontoclasts. MATERIALS AND METHODS: The maxillary first molars of 10-week-old male Wistar rats were moved mesially by a closed-coil spring for 14 days. Lithium chloride (LiCl; 0.64 mM/kg) or saline (control) was administered intraperitoneally daily. Tooth movements were measured using micro-computed tomography. Appearances of cell death, hyalinization, and odontoclasts were evaluated by histological analysis. RESULTS: OIRR observed on day 14 in the control group was suppressed strongly by LiCl administration. Apoptotic cells observed on day 1 in the compression area were gradually diminished on days 2 and 3 and transformed to hyalinization tissue in the control group. LiCl administration remarkably suppressed this cell death and subsequent hyalinization. Also, the appearance of odontoclasts in the compression area observed on day 7 was significantly suppressed by LiCl administration. Accordingly, these degenerative processes to OIRR were suppressed substantially by LiCl treatment. CONCLUSIONS: Lithium reduces OIRR through the suppression of periodontal ligament cell death, hyalinization, and odontoclast formation.

Runt-related transcription factor-2 (Runx2) is required for bone matrix protein gene expression in committed osteoblasts in mice.

Runt-related transcription factor-2 (Runx2) is an essential transcription factor for osteoblast differentiation. However, its functions after the commitment into osteoblasts are controversial and remain to be clarified. We generated enhanced green fluorescent protein (EGFP)-Cre transgenic mice driven by the 2.3-kilobase (kb) Col1a1 promoter, and Runx2 was deleted in osteoblasts and odontoblasts in Runx2fl/flCre mice. The sutures and fontanelles were more widely opened in Runx2fl/flCre newborns than in Runx2fl/fl newborns. Runx2fl/flCre mice exhibited dwarfism with shorter incisors and 37% had irregularly aligned incisors. The volume of trabecular bone in femurs and vertebrae and their bone mineral density (BMD), in addition to the cortical thickness and BMD were reduced in Runx2fl/flCre mice compared with Runx2fl/fl mice in both sexes. The bone formation of both trabecular and cortical bone, osteoblast number, osteoclast surface, osteoblast proliferation, and the serum levels of procollagen type 1 N-terminal propeptide (P1NP), tartrate-resistant acid phosphatase 5b (TRAP5b), and C-terminal cross-linked telopeptide of type 1 collagen (CTX1) were reduced in Runx2fl/flCre mice. The expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap&Bglap2, and of Tnfsf11 was lower in Runx2fl/flCre mice than in Runx2fl/fl mice. The expression of Runx2 target genes, including Ihh, Fgfr1, Fgfr2, Fgfr3, Tcf7, Wnt10b, Pth1r, Sp7, and Dlx5, was also reduced. Osteoblasts in Runx2fl/fl mice were cuboidal and contained abundant type I collagen α1 (Col1a1), whereas those in Runx2fl/flCre mice were deflated and contained a small amount of Col1a1. Runx2 activated the reporter activity of the 2.3-kb Col1a1 promoter and bound the region around the Col1a1 transcription start site. The deletion of Runx2 by Cre-expressing adenovirus in Runx2fl/fl primary osteoblasts impaired osteoblast differentiation and the expression of genes encoding major bone matrix proteins, and osteoclastogenesis was inhibited due to the reduction of Tnfsf11 expression in the osteoblasts. This study demonstrated that Runx2 is required for the expression of the major bone matrix protein genes and Tnfsf11 after commitment into osteoblasts in mice. © 2021 American Society for Bone and Mineral Research (ASBMR).

Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts.

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice.

Osteocalcin is necessary for the alignment of apatite crystallites, but not glucose metabolism, testosterone synthesis, or muscle mass.

The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.

Suppressive effects of N-bisphosphonate in osteoblastic cells mitigated by non-N-bisphosphonate but not by sodium-dependent phosphate cotransporter inhibitor.

There are two types of bisphosphonates (BPs), nitrogen-containing (N-BPs) and those free from nitrogen (non-N-BPs). Although N-BPs show greater inhibition of bone resorption than non-N-BPs, their effects are likely accompanied with inflammation, which non-N-BPs mitigate. We examined the competitive effects of zoledronate (ZOL), an N-BP, and etidronate (ETI), a non-N-BP, in osteoblasts. ZOL, but not ETI, markedly reduced alkaline phosphatase activity and cell viability in osteoblastic MC3T3-E1 and Saos2 cells, while that inhibition was relieved by simultaneous administration of ETI, possibly because of competition with ZOL for cellular uptake. However, phosphonoformate, an inhibitor of the phosphonate transporters SLC20A and SLC34A, did not mitigate the reducing effects of ZOL, suggesting that those transporters are not involved in BP uptake in osteoblastic cells. Additionally, ZOL reduced fibroblastic NIH3T3 and C3H10T1/2 cell viability, which was relieved by administration of both ETI and phosphonoformate. Transporter gene expression levels were significantly lower in osteoblasts as compared with fibroblasts, which may account for the distinct effects of phosphonoformate with different cell types. Together, our results suggest existence of a common uptake route of N-BPs and non-N-BPs into osteoblastic cells that is unrelated to the SLC20A and SLC34A families. SIGNIFICANCE OF THE STUDY: N-BP ZOL was shown to suppress differentiation and viability of osteoblasts. ZOL-induced cell viability suppression was also observed in fibroblasts, which was markedly relieved by addition of the non-N-BP ETI. Additionally, mitigation of the effects of ZOL was achieved with phosphonoformate, a sodium-phosphate cotransporter inhibitor, in fibroblastic cells but not osteoblasts. Expression levels of SLC20A and SLC34A family genes were significantly lower in osteoblasts as compared with fibroblasts. These observations suggest that incorporation of N-BPs and non-N-BPs in osteoblasts is mediated via common transporters that appear to be distinct from SLC20A and 34A, which operate in fibroblasts.

Runx2 regulates cranial suture closure by inducing hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in suture mesenchymal cells.

Cleidocranial dysplasia (CCD, #119600), which is characterized by hypoplastic clavicles, open fontanelles, supernumerary teeth and a short stature, is caused by heterozygous mutations in RUNX2. However, it currently remains unclear why suture closure is severely impaired in CCD patients. The closure of posterior frontal (PF) and sagittal (SAG) sutures was completely interrupted in Runx2+/- mice, and the proliferation of suture mesenchymal cells and their condensation were less than those in wild-type mice. To elucidate the underlying molecular mechanisms, differentially expressed genes between wild-type and Runx2+/- PF and SAG sutures were identified by microarray and real-time reverse transcription polymerase chain reaction analyses. The expression of hedgehog, Fgf, Wnt and Pthlh signaling pathway genes, including Gli1, Ptch1, Ihh, Fgfr2, Fgfr3, Tcf7, Wnt10b and Pth1r, which were directly regulated by Runx2, was reduced in the sutures, but not the calvarial bone tissues of Runx2+/- mice. Bone formation and suture closure were enhanced in an organ culture of Runx2+/- calvariae with ligands or agonists of hedgehog, Fgf, Wnt and Pthlh signaling, while they were suppressed and suture mesenchymal cell proliferation was decreased in an organ culture of wild-type calvariae with their antagonists. These results indicate that more than a half dosage of Runx2 is required for the proliferation of suture mesenchymal cells, their condensation and commitment to osteoblast-lineage cells, and the induction of hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in sutures, but not in calvarial bone tissues, and also that the activation of hedgehog, Fgf, Wnt and Pthlh signaling pathways is necessary for suture closure.

Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation by regulating Fgfr2 and Fgfr3.

Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1-3 regulation by Runx2. To investigate the physiological role of Runx2 in the regulation of Fgfr1-3, we compared osteoblast progenitors in Sp7-/- and Runx2-/- mice. Osteoblast progenitors accumulated and actively proliferated in calvariae and mandibles of Sp7-/- but not of Runx2-/- mice, and the number of osteoblast progenitors and their proliferation were dependent on the gene dosage of Runx2 in Sp7-/- background. The expression of Fgfr2 and Fgfr3, which were responsible for the proliferation of osteoblast progenitors, was severely reduced in Runx2-/- but not in Sp7-/- calvariae. Runx2 directly regulated Fgfr2 and Fgfr3, increased the proliferation of osteoblast progenitors, and augmented the FGF2-induced proliferation. The proliferation of Sp7-/- osteoblast progenitors was enhanced and strongly augmented by FGF2, and Runx2 knockdown reduced the FGF2-induced proliferation. Fgfr inhibitor AZD4547 abrogated all of the enhanced proliferation. These results indicate that Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation, at least partly, by regulating Fgfr2 and Fgfr3 expression.

Collapsin Response Mediator Protein 1, a Novel Marker Protein for Differentiated Odontoblasts.

We previously reported that the terminal differentiation of odontoblasts was inhibited in Runx2 transgenic {Tg(Col1a1-Runx2)} mice under the control of the 2.3-kb Col1a1 promoter. Odontoblasts in Tg(Col1a1-Runx2) mice lose their characteristic long cellular processes, and show marked reductions in the protein levels of markers for odontoblasts, such as dentin sialophosphoprotein, nestin, and microtubule-associated protein tau (Mapt). We herein demonstrated that collapsin response mediator protein 1 (CRMP1), a neuronal phosphoprotein that participates in various aspects of neuronal development, was specifically expressed in the differentiated odontoblasts of wild-type, but not Tg(Col1a1-Runx2) tooth germs by comparing expression profiles in wild-type and Tg(Col1a1-Runx2) mouse molars using microarray and immunohistochemical analyses. CRMP1 expression was detected at a slightly later differentiation stage in odontoblasts than type 1 collagen, nestin, and Mapt expression, which was observed from the onset of dentinogenesis. Among these proteins, CRMP1 was the most specifically localized in odontoblasts in the tooth germ. In erupted molars, odontoblast-specific CRMP1 expression decreased with age. These results indicate that CRMP1 is a novel marker protein for differentiated odontoblasts in mouse tooth germs, and suggest that CRMP1 participates in the morphogenesis of functioning odontoblasts.

Dental Calculus Stimulates Interleukin-1ß Secretion by Activating NLRP3 Inflammasome in Human and Mouse Phagocytes.

Dental calculus is a mineralized deposit associated with periodontitis. The bacterial components contained in dental calculus can be recognized by host immune sensors, such as Toll-like receptors (TLRs), and induce transcription of proinflammatory cytokines, such as IL-1ß. Studies have shown that cellular uptake of crystalline particles may trigger NLRP3 inflammasome activation, leading to the cleavage of the IL-1ß precursor to its mature form. Phagocytosis of dental calculus in the periodontal pocket may therefore lead to the secretion of IL-1ß, promoting inflammatory responses in periodontal tissues. However, the capacity of dental calculus to induce IL-1ß secretion in human phagocytes has not been explored. To study this, we stimulated human polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) with dental calculus collected from periodontitis patients, and measured IL-1ß secretion by ELISA. We found that calculus induced IL-1ß secretion in both human PMNs and PBMCs. Calculus also induced IL-1ß in macrophages from wild-type mice, but not in macrophages from NLRP3- and ASC-deficient mice, indicating the involvement of NLRP3 and ASC. IL-1ß induction was inhibited by polymyxin B, suggesting that LPS is one of the components of calculus that induces pro-IL-1ß transcription. To analyze the effect of the inorganic structure, we baked calculus at 250°C for 1 h. This baked calculus failed to induce pro-IL-1ß transcription. However, it did induce IL-1ß secretion in lipid A-primed cells, indicating that the crystalline structure of calculus induces inflammasome activation. Furthermore, hydroxyapatite crystals, a component of dental calculus, induced IL-1ß in mouse macrophages, and baked calculus induced IL-1ß in lipid A-primed human PMNs and PBMCs. These results indicate that dental calculus stimulates IL-1ß secretion via NLRP3 inflammasome in human and mouse phagocytes, and that the crystalline structure has a partial role in the activation of NLRP3 inflammasome.

Overexpression of BCLXL in Osteoblasts Inhibits Osteoblast Apoptosis and Increases Bone Volume and Strength.

The Bcl2 family proteins, Bcl2 and BclXL, suppress apoptosis by preventing the release of caspase activators from mitochondria through the inhibition of Bax subfamily proteins. We reported that BCL2 overexpression in osteoblasts using the 2.3 kb Col1a1 promoter increased osteoblast proliferation, failed to reduce osteoblast apoptosis, inhibited osteoblast maturation, and reduced the number of osteocyte processes, leading to massive osteocyte death. We generated BCLXL (BCL2L1) transgenic mice using the same promoter to investigate BCLXL functions in bone development and maintenance. Bone mineral density in the trabecular bone of femurs was increased, whereas that in the cortical bone was similar to that in wild-type mice. Osteocyte process formation was unaffected and bone structures were similar to those in wild-type mice. A micro-CT analysis showed that trabecular bone volume in femurs and vertebrae and the cortical thickness of femurs were increased. A dynamic bone histomorphometric analysis revealed that the mineralizing surface was larger in trabecular bone, and the bone-formation rate was increased in cortical bone. Serum osteocalcin but not TRAP5b was increased, BrdU-positive osteoblastic cell numbers were increased, TUNEL-positive osteoblastic cell numbers were reduced, and osteoblast marker gene expression was enhanced in BCLXL transgenic mice. The three-point bending test indicated that femurs were stronger in BCLXL transgenic mice than in wild-type mice. The frequency of TUNEL-positive primary osteoblasts was lower in BCLXL transgenic mice than in wild-type mice during cultivation, and osteoblast differentiation was enhanced but depended on cell density, indicating that enhanced differentiation was mainly owing to reduced apoptosis. Increased trabecular and cortical bone volumes were maintained during aging in male and female mice. These results indicate that BCLXL overexpression in osteoblasts increased the trabecular and cortical bone volumes with normal structures and maintained them majorly by preventing osteoblast apoptosis, implicating BCLXL as a therapeutic target of osteoporosis. © 2016 American Society for Bone and Mineral Research.

Microtubule-associated protein tau (Mapt) is expressed in terminally differentiated odontoblasts and severely down-regulated in morphologically disturbed odontoblasts of Runx2 transgenic mice.

Runx2 is an essential transcription factor for osteoblast and odontoblast differentiation and the terminal differentiation of chondrocytes. We have previously shown that the terminal differentiation of odontoblasts is inhibited in Runx2 transgenic {Tg(Col1a1-Runx2)} mice under the control of the 2.3-kb Col1a1 promoter, which directs the transgene expression to osteoblasts and odontoblasts. Odontoblasts show severe reductions in Dspp and nestin expression and lose their characteristic polarized morphology, including a long process extending to dentin, in Tg(Col1a1-Runx2) mice. We study the molecular mechanism of odontoblast morphogenesis by comparing gene expression in the molars of wild-type and Tg(Col1a1-Runx2) mice, focusing on cytoskeleton-related genes. Using microarray, we found that the gene expression of microtubule-associated protein tau (Mapt), a neuronal phosphoprotein with important roles in neuronal biology and microtubule dynamics and assembly, was high in wild-type molars but severely reduced in Tg(Col1a1-Runx2) molars. Immunohistochemical analysis revealed that Mapt was specifically expressed in terminally differentiated odontoblasts including their processes in wild-type molars but its expression was barely detectable in Tg(Col1a1-Runx2) molars. Double-staining of Mapt and Runx2 showed their reciprocal expression in odontoblasts. Mapt and tubulin co-localized in odontoblasts in wild-type molars. Immunoelectron microscopic analysis demonstrated Mapt lying around α-tubulin-positive filamentous structures in odontoblast processes. Thus, Mapt is a useful marker for terminally differentiated odontoblasts and might play an important role in odontoblast morphogenesis.

Carbenoxolone-sensitive and cesium-permeable potassium channel in the rod cells of frog taste discs.

The rod cells in frog taste discs display the outward current and maintain the negative resting potential in the condition where internal K+ is replaced with Cs+. We analyzed the properties of the Cs+-permeable conductance in the rod cells. The current-voltage (I/V) relationships obtained by a voltage ramp were bell-shaped under Cs+ internal solution. The steady state I/V relationships elicited by voltage steps also displayed the bell-shaped outward current. The activation of the current accelerated with the depolarization and the inactivation appeared at positive voltage. The gating for the current was maintained even at symmetric condition (Cs+ external and internal solutions). The wing cells did not show the properties. The permeability for K+ was a little larger than that for Cs+. Internal Na+ and NMDG+ could not induce the bell-shaped outward current. Carbenoxolone inhibited the bell-shaped outward Cs+ current dose dependently (IC50 : 27 µM). Internal arachidonic acid (20 µM) did not induce the linear current-voltage (I-V) relationship which is observed in two-pore domain K+ channel (K2P). The results suggest that the resting membrane potentials in the rod cells are maintained by the voltage-gated K+ channels.

Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans.

Galnt3, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-D-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2(-/-) cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3(-/-) mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3(-/-) mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation.

Dlx5 and mef2 regulate a novel runx2 enhancer for osteoblast-specific expression.

Runx2 is essential for osteoblast differentiation and chondrocyte maturation. The expression of Runx2 is the first requisite step for the lineage determination from mesenchymal stem cells to osteoblasts. Although the transcript from Runx2 distal promoter is majorly expressed in osteoblasts, the promoter failed to direct green fluorescent protein (GFP) expression to osteoblasts. To find the regulatory region, we generated GFP reporter mice driven by a bacterial artificial chromosome (BAC) of Runx2 locus, and succeeded in the reproduction of endogenous Runx2 expression. By serially deleting it, we identified a 343-bp enhancer, which directed GFP expression specifically to osteoblasts, about 30 kb upstream of the distal promoter. The sequence of the 343-bp enhancer was highly conserved among mouse, human, dog, horse, opossum, and chicken. Dlx5, Mef2c, Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, which localized on the enhancer region in primary osteoblasts, synergistically upregulated the enhancer activity, whereas Msx2 downregulated the activity in mouse osteoblastic MC3T3-E1 cells. Msx2 was predominantly bound to the enhancer in mouse multipotent mesenchymal C3H10T1/2 cells, whereas Dlx5 was predominantly bound to the enhancer in MC3T3-E1 cells. Dlx5 and Mef2 directly bound to the enhancer, and the binding sites were required for the osteoblast-specific expression in mice, whereas the other factors bound to the enhancer by protein-protein interaction. The enhancer was characterized by the presence of the histone variant H2A.Z, the enrichment of histone H3 mono- and dimethylated at Lys4 and acetylated at Lys18 and Lys27, but the depletion of histone H3 trimethylated at Lys4 in primary osteoblasts. These findings indicated that the enhancer, which had typical histone modifications for enhancers, contains sufficient elements to direct Runx2 expression to osteoblasts, and that Dlx5 and Mef2, which formed an enhanceosome with Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, play an essential role in the osteoblast-specific activation of the enhancer. © 2014 American Society for Bone and Mineral Research.

Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements.

Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2, as well as Cfm1/Cfm2 double-knockout (Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb)-deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics.

Wing (Ib) cells in frog taste discs detect dietary unsaturated fatty acids.

The effects of unsaturated fatty acids on membrane properties were studied using conventional whole-cell patch-clamp recording of isolated wing (Ib) cells in bullfrog (Lithobates catesbeianus) taste discs. Applying arachidonic acid to the bath induced monophasic inward currents in 60% of wing cells and biphasic inward and outward currents in the other cells. The intracellular dialysis of arachidonic acid did not induce an inward current; however, it enhanced a slowly developing Ba(2+)-sensitive outward current. The effects of various unsaturated fatty acids were explored under the condition of Cs(+) internal solution. Linoleic and α-linolenic acids induced large inward currents. Oleic, eicosapentaenoic and docosahexaenoic acids elicited the same inward currents as those of arachidonic acid. Wing cells, under the basal condition with Cs(+) internal solution, displayed a small inward current of -1.1±0.1pA/pF at -50mV (n=40), in which the peak existed at a membrane potential of -49mV. Removing external Ca(2+) further increased the inward current by -2.9±0.3pA/pF at -50mV (n=4) from the basal current and the peak was located at -55mV. External linoleic acid (50µM) also induced a similar inward current of -5.6±0.6pA/pF at -50mV (n=19) from the basal current and the peak was located at -61mV. External Ca(2+)-free saline and linoleic acid induced similar current/voltage (I/V) relationships elicited by a ramp voltage as well as voltage steps. Linoleic acid-induced currents were not influenced by replacing internal EGTA with BAPTA, whereas inward currents disappeared under the elimination of external Na(+) and addition of flufenamic acid. These results suggest that dietary unsaturated fatty acids may depolarize wing (Ib) cells, which affects the excitability of these cells.

Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia.

Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3(-/-)) mice were infertile, as previously reported by Ichikawa et al. (2009). To investigate the involvement of Galnt3 in spermatogenesis, we examined the differentiation of germ cells in Galnt3(-/-) mice. Galnt3 mRNA was most highly expressed in testis, and Galnt3 protein was localized in the cis-medial parts of the Golgi stacks of spermatocytes and spermatids in the seminiferous tubules. Spermatozoa in Galnt3(-/-) mice were rare and immotile, and most of them had deformed round heads. They exhibited abnormal acrosome and disturbed mitochondria arrangement in the flagella. At the cap phase, proacrosomal vesicles of various sizes, which had not coalesced to form a single acrosomal vesicle, were attached to the nucleus in Galnt3(-/-) mice. TUNEL-positive cells were increased in the seminiferous tubules. The binding of VVA lectin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), in the acrosomal regions of spermatids and spermatozoa in Galnt3(-/-) mice was drastically reduced. Equatorin is a N, O-sialoglycoprotein localized in the acrosomal membrane and is suggested to be involved in sperm-egg interaction. Immunohistochemical and Western blot analyses showed a drastic reduction in the reactivity with MN9 antibody, which recognizes the O-glycosylated moiety of equatorin and inhibits sperm-egg interaction. These findings indicate that deficiency of Galnt3 results in a severe reduction of mucin-type O-glycans in spermatids and causes impaired acrosome formation, leading to oligoasthenoteratozoospermia, and suggest that Galnt3 may also be involved in the process of fertilization through the O-glycosylation of equatorin.

Osteocyte network; a negative regulatory system for bone mass augmented by the induction of Rankl in osteoblasts and Sost in osteocytes at unloading.

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.

SP7 inhibits osteoblast differentiation at a late stage in mice.

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2(-/-) calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter.

Pyruvate dehydrogenase kinase 4 induces bone loss at unloading by promoting osteoclastogenesis.

Disuse osteoporosis, which occurs commonly in prolonged bed rest and immobilization, is becoming a major problem in modern societies; however, the molecular mechanisms underlying unloading-driven bone loss have not been fully elucidated. The osteocyte network is considered to be an ideal mechanosensor and mechanotransduction system. We searched for the molecules responsible for disuse osteoporosis using BCL2 transgenic mice, in which the osteocyte network was disrupted. Pyruvate dehydrogenase kinase 4 (Pdk4), which inactivates pyruvate dehydrogenase complex (PDC), was upregulated in femurs and tibiae of wild-type mice but not of BCL2 transgenic mice after tail suspension. Bone in Pdk4(-/-) mice developed normally and was maintained. At unloading, however, bone mass was reduced due to enhanced osteoclastogenesis and Rankl expression in wild-type mice but not in Pdk4(-/-) mice. Osteoclast differentiation of Pdk4(-/-) bone marrow-derived monocyte/macrophage lineage cells (BMMs) in the presence of M-CSF and RANKL was suppressed, and osteoclastogenesis was impaired in the coculture of wild-type BMMs and Pdk4(-/-) osteoblasts, in which Rankl expression and promoter activity were reduced. Further, introduction of Pdk4 into Pdk4(-/-) BMMs and osteoblasts enhanced osteoclastogenesis and Rankl expression and activated Rankl promoter. These findings indicate that Pdk4 plays an important role in bone loss at unloading by promoting osteoclastogenesis.