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Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase‐dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14‐Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red‐positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis. [ABSTRACT FROM AUTHOR]

ClC-7 [Cl.sup.-] channels expressed in osteoclasts are important for bone resorption since it has been shown that disruption of the ClCN7 gene in mice leads to severe osteopetrosis. We have previously reported that [Cl.sup.-] currents recorded from mouse osteoclasts resemble those of ClC-3 [Cl.sup.-] channels. The aim of the present study was to determine the expression of ClC-3 channels in mouse osteoclasts and their functional role during bone resorption. We detected transcripts for both CIC-7 and CIC-3 channels in mouse osteoclasts by RT-PCR. The expression of ClC-3 was confirmed by immunocytochemical staining. Mouse osteoclasts lacking ClC-3 [Cl.sup.-] channels (Cl[C-3.sup.-/-] osteoclasts) derived from ClCN3 gene-deficient mice (Cl[C-3.sup.-/-]) showed lower bone resorption activity compared with Cl[C-3.sup.+/+] osteoclasts derived from wild-type mice (Cl[C-3.sup.+/+]). Treatment of Cl[C-3.sup.+/+] osteoclasts with small interfering RNA (siRNA) against ClC-3 also significantly reduced bone resorption activity. Electrophysiological properties of basal and hypotonicity-induced [Cl.sup.-] currents in Cl[C-3.sup.-/-] osteoclasts did not differ significantly from those in Cl[C-3.sup.+/+] osteoclasts. Using immunocytochemistry, ClC-3 was colocalized with lysosome-associated membrane protein 2. Using pH-sensitive dyes, organelle acidification activity in Cl[C-3.sup.-/-] osteoclasts was weaker than in Cl[C-3.sup.+/+] osteoclasts. Treatment of Cl[C-3.sup.+/+] osteoclasts with siRNA against ClC-3 also reduced the organelle acidification activity. In conclusion, ClC-3 [Cl.sup.-] channels are expressed in intracellular organelles of mouse osteoclasts and contribute to osteoclastic bone resorption in vitro through organelle acidification. knockout mice; [Cl.sup.-] current; lysosome-associated membrane protein; pH-sensitive dye staining

We examined changes in electrical and morphological properties of rat osteoclasts in response to prostaglandin (PG)[E.sub.2]. PG[E.sub.2] (> 10 nM) stimulated an outwardly rectifying [Cl.sup.-] current in a concentration-dependent manner and caused a long-lasting depolarization of cell membrane. This PG[E.sub.2]-induced [Cl.sup.-] current was reversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and tamoxifen. The anion permeability sequence of this current was [I.sup.-] > [Br.sup.-][approximately equal to] [Cl.sup.-] > [gluconate.sup.-]. When outwardly rectifying [Cl.sup.-] current was induced by hyposmotic extracellular solution, no further stimulatory effect of PG[E.sub.2] was seen. Forsknlin and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) mimicked the effect of PG[E.sub.2]. The PG[E.sub.2]-induced [Cl.sup-] current was inhibited by pretreatment with guanosine 5'-O-2-(thiodiphosphate) (GDP[beta]S), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS), N-(2-[p-bromocinnmnylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), and protein kinase A inhibitors. Even in the absence of nonosteoclastic cells, PG[E.sub.2] (1 [micro]M) reduced cell surface area and suppressed motility of osteoclasts, and these effects were abolished by Rp-cAMPS or H-89. PG[E.sub.2] is known to exert its effects through four subtypes of PGE receptors (EP1-EP4). EP2 and EP4 agonists (ONO-AE1-259 and ONO-AE1-329, respectively), but not EP1 and EP3 agonists (ONO-DI-004 and ONO-AE-248, respectively), mimicked the electrical and morphological actions of PG[E.sub.2] on osteoclasts. Our results show that PG[E.sub.2] stimulates rat osteoclast [Cl.sup.-] current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption. prostanoid receptor agonists; electrophysiology; motile activity; bone resorption

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis and periodontitis. Inflammation-induced bone loss of this sort results from elevated numbers of bone-resorbing osteoclasts. Gene targeting studies have shown that the transcription factor nuclear factor-[kappa]B (NF-[kappa]B) has a crucial role in osteoclast differentiation, and blocking NF-[kappa]B is a potential strategy for preventing inflammatory bone resorption. We tested this approach using a cell-permeable peptide inhibitor of the I[kappa]B-kinase complex, a crucial component of signal transduction pathways to NF-[kappa]B. The peptide inhibited RANKL-stimulated NF-[kappa]B activation and osteoclastogenesis both in vitro and in vivo. In addition, this peptide significantly reduced the severity of collagen-induced arthritis in mice by reducing levels of tumor necrosis factor-[alpha] and interleukin-1[beta], abrogating joint swelling and reducing destruction of bone and cartilage. Therefore, selective inhibition of NF-[kappa]B activation offers an effective therapeutic approach for inhibiting chronic inflammatory diseases involving bone resorption., Author(s): Eijiro Jimi [1, 5]; Kazuhiro Aoki [2]; Hiroaki Saito [2]; Fulvio D'Acquisto [1]; Michael J May [1, 6]; Ichiro Nakamura [3]; Testuo Sudo [4]; Takefumi Kojima [2]; Fujio Okamoto [...]

Osteoclasts are multinucleated, bone-resorbing cells that show structural and functional differences between the resorbing and nonresorbing (motile) states during the bone resorption cycle. In the present study, we measured intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in nonresorbing vs. resorbing rat osteoclasts. Basal [[[Ca.sup.2+]].sub.i] in osteoclasts possessing pseudopodia (nonresorbing/motile state) was around 110 nM and significantly higher than that in actin ring-forming osteoclasts (resorbing state, around 50 nM). In nonresorbing/motile osteoclasts, exposure to high [K.sup.+] reduced [[[Ca.sup.2+]].sub.i], whereas high [K.sup.+] increased [[[Ca.sup.2+]].sub.i] in resorbing state osteoclasts. In nonresorbing/motile cells, membrane depolarization and hyperpolarization applied by the patch-clamp technique decreased and increased [[[Ca.sup.2+]].sub.i], respectively. Removal of extracellular [Ca.sup.2+] or application of 300 [micro]M [La.sup.3+] reduced [[[Ca.sup.2+]].sub.i] to ~50 nM in non-resorbing/motile osteoclasts, and high-[K.sup.+]-induced reduction of [[[Ca.sup.2+]].sub.i] could not be observed under these conditions. Neither inhibition of intracellular [Ca.sup.2+] stores or plasma membrane [Ca.sup.2+] pumps nor blocking of L- and N-type [Ca.sup.2+] channels significantly reduced [[[Ca.sup.2+]].sub.i]. Exposure to high [K.sup.+] inhibited the motility of nonresorbing osteoclasts and reduced the number of actin rings and pit formation in resorbing osteoclasts. These results indicate that in nonresorbing/ motile osteoclasts, a [La.sup.3+]-sensitive [Ca.sup.2+] entry pathway is continuously active under resting conditions, keeping [[[Ca.sup.2+]].sub.i] high. Changes in membrane potential regulate osteoclastic motility by controlling the net amount of [Ca.sup.2+] entry in a 'reversed' voltage-dependent manner, i.e., depolarization decreases and hyperpolarization increases [[[Ca.sup.2+]].sub.i]. membrane depolarization; resorbing and motile activities; bone resorbing cycle

We investigated the transport of salicylic acid and L-lactic acid across the placenta using the human trophoblast cell line BeWo. We performed uptake experiments and measured the change in intracellular pH ([pH.sup.i]). The uptakes of [[sup.14]C] salicylic acid and L-[[sup.14]C]lactic acid were temperature- and extracellular pH-dependent and saturable at higher concentrations. Both uptakes were also reduced by FCCP, nigericin, and NA[N.sub.3]. Various nonsteroidal anti-inflammatory drugs (NSAIDs) strongly inhibited the uptake of L-[[sup.14]C]lactic acid. Salicylic acid and ibuprofen noncompetitively inhibited the uptake of L-[[sup.14]C]lactic acid. [alpha]-Cyano-4-hydroxycinnamate (CHC), a monocarboxylate transporter inhibitor, suppressed the uptake of L-[[sup.14]C]lactic acid but not that of [[sup.14]C]salicylic acid. CHC also suppressed the decrease of [pH.sub.i] induced by L-lactic acid but had little effect on that induced by salicylic acid or diclofenac. These results suggest that NSAIDs are potent inhibitors of lactate transporters, although they are transported mainly by a transport system distinct from that for L-lactic acid. L-lactic acid; blood-placental barrier; monocarboxylate transporter; [alpha]-cyano-4-hydroxycinnamate; intracellular pH

Programmed cell death ligands (PD‐Ls) are expressed in tumor cells where they bind to programmed cell death‐1, an immunocyte co–receptor, resulting in tumor cell evasion from the immune system. Chemotherapeutic drugs have been recently reported to induce the expression of PD‐L, such as PD‐L1, in some cancer cells. However, little is known regarding PD‐L2 expression and its role in oral squamous cell carcinoma (OSCC). In this study, we examined the effect of cisplatin on the expression and regulation of PD‐L2 in OSCC cell lines and analyzed malignant behavior in PD‐L2‐expressing cells using colony, transwell and transformation assays. In addition, we examined PD‐L2 expression in the tumor tissues of OSCC patients using cytology and tissue microarray methods. In OSCC cell lines, cisplatin treatment upregulated PD‐L2 expression, along with that of the drug efflux transporter ABCG2, via signal transducers and activator of transcription (STAT) 1/3 activation. Moreover, PD‐L2‐positive or PD‐L2‐overexpressing cells demonstrated upregulation in both invasion and transformation ability but not in proliferation compared with PD‐L2‐negative or PD‐L2‐silencing cells. PD‐L2 expression was also observed in OSCC cells of cytology samples and tissue from OSCC patients. The intensity of PD‐L2 expression was correlated with more malignant morphological features in the histological appearance and an invasive pattern. Our findings indicate that cisplatin‐upregulated PD‐L2 expression in OSCC via STAT1/3 activation and the expression of PD‐L2 are likely to be associated with malignancy in OSCC. The PD‐L2 expression in cisplatin‐resistant OSCC cells may be a critical factor in prognosis of advanced OSCC patients. [ABSTRACT FROM AUTHOR]

Scaffolds implanted into bone defect sites must achieve optimal biodegradation rates while appropriately filling the void as new bone formation progresses. We recently developed a unique biomaterial consisting of salmon deoxyribose nucleic acid (DNA) and protamine, which can be used as an osteoconductive scaffold for tissue engineering. The aim of the present study was to elucidate how the degradation rate of the scaffold affects bone regeneration. We examined the relationships between the degradation rate of salmon DNA scaffolds and new bone formation using a rat skin flank subcutaneous model and rat calvarial defect model. The degradation rates of the scaffolds were proportional to the durations of pretreatment with ultraviolet (UV) light irradiation. The biodegradation rates of the scaffolds were also dependent on the duration of UV irradiation, as tested a subcutaneous tissue implantation. Scaffolds irradiated with UV light for 0.5 h maintained gradual biodegradation of phosphate compared with scaffolds irradiated for 0 or 3 h. In the calvarial defect model, we found that new bone formation was higher in rats treated with scaffolds irradiated with UV light for 0.5 h compared with those irradiated with UV light for 0 or 3.0 h. The present results suggest that bioengineering of scaffolds for biodegradation is important to regenerate bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 122–128, 2019. [ABSTRACT FROM AUTHOR]

Rab44 is an atypical Rab GTPase that contains some additional domains such as the EF-hand and coiled-coil domains as well as Rab-GTPase domain. Although Rab44 genes have been found in mammalian genomes, no studies concerning Rab44 have been reported yet. Here, we identified Rab44 as an upregulated protein during osteoclast differentiation. Knockdown of Rab44 by small interfering RNA promotes RANKL-induced osteoclast differentiation of the murine monocytic cell line, RAW-D or of bone marrow-derived macrophages (BMMs). In contrast, overexpression of Rab44 prevents osteoclast differentiation. Rab44 was localized in the Golgi complex and lysosomes, and Rab44 overexpression caused an enlargement of early endosomes. A series of deletion mutant studies of Rab44 showed that the coiled-coil domain and lipidation sites of Rab44 is important for regulation of osteoclast differentiation. Mechanistically, Rab44 affects nuclear factor of activated T-cells c1 (NFATc1) signaling in RANKL-stimulated macrophages. Moreover, Rab44 depletion caused an elevation in intracellular Ca transients upon RANKL stimulation, and particularly regulated lysosomal Ca influx. Taken together, these results suggest that Rab44 negatively regulates osteoclast differentiation by modulating intracellular Ca levels followed by NFATc1 activation. [ABSTRACT FROM AUTHOR]

Folliculin-interacting protein 1 and 2 (FNIP1 and FNIP2) play critical roles in preventing renal malignancy through their association with the tumor suppressor FLCN. Mutations in FLCN are associated with Birt-Hogg-Dubé (BHD) syndrome, a rare disorder with increased risk of renal cancer. Recent studies indicated that FNIP1/FNIP2 double knockout mice display enlarged polycystic kidneys and renal carcinoma, which phenocopies FLCN knockout mice, suggesting that these two proteins function together to suppress renal cancer. However, the molecular mechanism functionally linking FNIP1/FNIP2 and FLCN remains largely elusive. Here, we demonstrated that FNIP2 protein is unstable and subjected to proteasome-dependent degradation via β-TRCP and Casein Kinase 1 (CK1)-directed ubiquitination in a nutrition-dependent manner. Degradation of FNIP2 leads to lysosomal dissociation of FLCN and subsequent lysosomal association of mTOR, which in turn promotes the proliferation of renal cancer cells. These results indicate that SCFβ-TRCP negatively regulates the FLCN complex by promoting FNIP degradation and provide molecular insight into the pathogenesis of BHD-associated renal cancer.

Osteoclast differentiation is critically dependent on calcium (Ca2+) signaling. Transient receptor potential vanilloid 4 (TRPV4), mediates Ca2+ influx in the late stage of osteoclast differentiation and thereby regulates Ca2+ signaling. However, the system-modifying effect of TRPV4 activity remains to be determined. To elucidate the mechanisms underlying TRPV4 activation based on osteoclast differentiation, TRPV4 gain-of-function mutants were generated by the amino acid substitutions R616Q and V620I in TRPV4 and were introduced into osteoclast lineage in Trpv4 null mice to generate Trpv4 R616Q/V620I transgenic mice. As expected, TRPV4 activation in osteoclasts increased the number of osteoclasts and their resorption activity, thereby resulting in bone loss. During in vitro analysis, Trpv4 R616Q/V620I osteoclasts showed activated Ca2+/calmodulin signaling compared with osteoclasts lacking Trpv4. In addition, studies of Trpv4 R616Q/V620I mice that lacked the calmodulin-binding domain indicated that bone loss due to TRPV4 activation was abrogated by loss of interactions between Ca2+/calmodulin signaling and TRPV4. Finally, modulators of TRPV4 interactions with the calmodulin-binding domain were investigated by proteomic analysis. Interestingly, nonmuscle myosin IIa was identified by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis, which was confirmed by immunoblotting following coimmunoprecipitation with TRPV4. Furthermore, myosin IIa gene silencing significantly reduced TRPV4 activation concomitant with impaired osteoclast maturation. These results indicate that TRPV4 activation reciprocally regulates Ca2+/calmodulin signaling, which involves an association of TRPV4 with myosin IIa, and promotes sufficient osteoclast function. © 2012 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

ClC7 Cl− channels ( Clcn7) are crucial for osteoclastic bone resorption and have heterozygous mutation in autosomal osteopetrosis type II (ADO II) patients. Although extracellular acidification is known to induce ClC7 Cl− currents in Clcn7-transfected oocytes, other characteristics of this acid-induced Cl− current, as well as the effects of mutant Clcn7 in ADO II, remain to be determined. The present study showed that extracellular acidification evoked outward Cl− currents in mouse osteoclasts. Expression of wild-type human Clcn7 in HEK293 cells also induced a significant increase in acid-activated Cl− currents. These acid-activated Cl− currents were independent of intracellular acidification and [Ca2+] i increase. HEK293 cells with the Clcn7 mutation associated with ADO II at G215R did not display these Cl− currents. These results suggest that osteoclastic ClC7 Cl− channels are activated under extracellar acidification and suppressed in Clcn7 mutant associated with ADO II during bone resorption. [ABSTRACT FROM AUTHOR]

Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective γ-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-κB pathways that is relevant in osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis and periodontitis. Inflammation-induced bone loss of this sort results from elevated numbers of bone-resorbing osteoclasts. Gene targeting studies have shown that the transcription factor nuclear factor-?B (NF-?B) has a crucial role in osteoclast differentiation, and blocking NF-?B is a potential strategy for preventing inflammatory bone resorption. We tested this approach using a cell-permeable peptide inhibitor of the I?B-kinase complex, a crucial component of signal transduction pathways to NF-?B. The peptide inhibited RANKL-stimulated NF-?B activation and osteoclastogenesis both in vitro and in vivo. In addition, this peptide significantly reduced the severity of collagen-induced arthritis in mice by reducing levels of tumor necrosis factor-a and interleukin-1ß, abrogating joint swelling and reducing destruction of bone and cartilage. Therefore, selective inhibition of NF-?B activation offers an effective therapeutic approach for inhibiting chronic inflammatory diseases involving bone resorption. [ABSTRACT FROM AUTHOR]

Osteoclasts are multinucleated, bone-resorbing cells that show structural and functional differences between the resorbing and nonresorbing (motile) states during the bone resorption cycle. In the present study, we measured intracellular Ca[sup 2+] concentration ([Ca[sup 2+]][sub i]) in nonresorbing vs. resorbing rat osteoclasts. Basal [Ca[sup 2+]][sub i] in osteoclasts possessing pseudopodia (nonresorbing/ motile state) was around 110 nM and significantly higher than that in actin ring-forming osteoclasts (resorbing state, around 50 nM). In nonresorbing/motile osteoclasts, exposure to high K[sup +] reduced [Ca[sup 2+]][sub i], whereas high K[sup +] increased [Ca[sup 2+]][sub i] in resorbing state osteoclasts. In nonresorbing/motile ceils, membrane depolarization and hyperpolarization appiled by the patch-clamp technique decreased and increased [Ca[sup 2+]][sub i], respectively. Removal of extracellular Ca[sup 2+] or application of 300 µM La[sup 3+] reduced [Ca[sup 2+][sub i] to ∼50 nM in nonresorbing/motile osteoclasts, and high-K[sup +]-induced reduction of [Ca[sup 2+]][sub i] could not be observed under these conditions. Neither inhibition of intracellular Ca[sup 2+] stores or plasma membrane Ca[sup 2+] pumps nor blocking of L- and N-type Ca[sup 2+] channels significantly reduced [Ca[sup 2+][sub i]. Exposure to high K[sup +] inhibited the motility of nonresorbing osteoclasts and reduced the number of actin rings and pit formation in resorbing osteoclasts. These results indicate that in nonresorbing/ motile osteoclasts, a La[sup 3+]-sensitive Ca[sup 2+] entry pathway is continuously active under resting conditions, keeping [Ca[sup 2+]][sub i] high. Changes in membrane potential regulate osteoclastic motility by controlling the net amount of Ca[sup 2+] entry in a "reversed" voltage-dependent manner, i.e., depolarization decreases and hyperpolarization increases [Ca[sup 2+]][sub i]. [ABSTRACT FROM AUTHOR]

Examines the transport of salicylic acid across the placenta using the human trophoblast cell line BeWo. Measurement of the changes in intracellular pH during acid uptakes; Inhibition of L-lactic acid by salicylic acid and ibuprofen; Correlation of nonsteroidal anti-inflammatory drugs with lactate transporter inhibition.

Using the whole-cell patch-clamp technique in freshly isolated rat osteoclasts we examined the effects of estrogen on ionic channels. The predominant current was an inward rectifier K+ current (IKir). In the absence of non-osteoclastic cells, extracellularly applied 17β-estradiol (>0.1 µM) inhibited IKir, indicating that estrogen acts directly on osteoclasts. Application of 17β-estradiol (10 µM) for 10 min reduced IKir at the membrane potential of –120 mV to 70±15% of control. Removal of 17β-estradiol partially restored the inhibition. The inhibition of IKir was dependent on concentration and application time. Intracellularly applied 17β-estradiol had no effect on IKir. 17α-Estradiol also inhibited the IKir, whereas progesterone and testosterone had no effect. The inhibitory action of 17β-estradiol was not affected by guanosine 5′-O-(2-thiodiphosphate) (GDPβS), adenosine 3′,5′-cyclic monophosphothioate Rp diastereomer (Rp-cAMPS), okadaic acid, staurosporine and phorbol ester, and was independent of intracellular Ca2+ concentration ([Ca2+]i). With no influence from soluble factors secreted from non-osteoclastic cells, preincubation of the osteoclasts for more than 60 min with much lower concentrations of 17β-estradiol (1 and 10 nM) caused a reduction of IKir. In current-clamp configuration, application of 17β-estradiol (10 µM) depolarized the membrane associated with a decrease in a membrane conductance, indicating that 17β-estradiol inhibits IKir and depolarizes the membrane of osteoclasts. These results suggest that the 17β-estradiol-induced inhibition of IKir might be mediated via non-genomic mechanisms. This direct action of 17β-estradiol on osteoclasts may contribute to the regulation of [Ca2+]i and partially account for the protective effects of estrogen against bone loss. [ABSTRACT FROM AUTHOR]