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IntroductionEstrogens inhibit bone resorption and preserve bone mass, at least in part, via direct effects on osteoclasts. The binding of RANKL, the critical cytokine for osteoclast differentiation, to its receptor in osteoclast precursor cells of the monocyte lineage recruits the adaptor protein TRAF6 and activates multiple signaling pathways. Early effects of RANKL include stimulation of mitochondria. 17β-estradiol (E2) prevents the effects of RANKL on mitochondria and promotes mitochondria mediated apoptotic cell death. However, the molecular mechanisms responsible for the actions of RANKL and estrogens on mitochondria remain unknown. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a complex I-associated protein that regulates immune responses in macrophages following the engagement of Toll-like receptors, which also recruit TRAF6. Here, we examined whether ECSIT could be implicated in the rapid effects of RANKL and E2 on osteoclast progenitors.MethodsBone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E2 (10-8 M). ECSIT-TRAF6 interaction was evaluated by co-immunoprecipitation and ECSIT levels in mitochondria and cytosolic fractions by Western blot. ShRNA lentivirus particles were used to knockdown ECSIT. Osteoclasts were enumerated after tartrate-resistant acid phosphatase staining. Oxygen consumption and extracellular acidification rates were measured with Seahorse XFe96 Analyzer. ATP, lactate, and NAD/NADH were measured with commercial assay kits. NADH oxidation to NAD was used to evaluate Complex I activity. Total and mitochondrial ROS, and mitochondrial membrane potential were measured with H2DCFDA, MitoSOX, and TMRM probes, respectively. Degradation of DEVD-AFC was used to measure Caspase-3 activity.ResultsWe found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E2 on osteoclastogenesis. Loss of ECSIT decreased complex I activity, oxygen consumption, NAD+/NADH redox ratio, and ATP production and increased mitochondrial ROS. In the absence of ECSIT, the stimulatory actions of RANKL on complex I activity and all other markers of oxidative phosphorylation, as well as their inhibition by E2, were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors.DiscussionThese findings suggest that dysregulated mitochondria cause a switch in RANKL signaling from pro-survival to pro-apoptotic. In addition, our results indicate that ECSIT represents a central node for the early effects of RANKL on mitochondria and that inhibition of ECSIT-mediated mitochondria stimulation might contribute to the bone protective actions of estrogens.

PDF file - 256K, Figure S1. Effects of aCXCL10 on tumor metastasis into lungs and adrenal glands. Figure S2. CXCL10-induced adhesion and survival of B16F10 cells

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PDF file - 110K, Primer sequences for real-time PCR and RT-PCR

Hem1 (Hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity, as well as for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass due to defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially due to decreased MAPK and tyrosine kinase c-Abl activity. Transplantation of bone marrow hematopoietic stem and progenitor cells from wild-type into Hem1 knockout mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.

The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal cells, but functional evidence for the mediators of these effects has only recently begun to emerge. We report that the matrix metalloproteinase 13 (MMP13) is the highest up-regulated gene in mesenchymal cells from mice lacking the estrogen receptor alpha (ERα). In sham-operated female mice with conditional Mmp13 deletion in Prrx1 expressing cells (Mmp13ΔPrrx1), the femur and tibia length was lower as compared to control littermates (Mmp13f./f). Additionally, in the sham-operated female Mmp13ΔPrrx1 mice cortical thickness and trabecular bone volume in the femur and tibia were higher and osteoclast number at the endocortical surfaces was lower, whereas bone formation rate was unaffected. Notably, the decrease of cortical thickness caused by ovariectomy (OVX) in the femur and tibia of Mmp13f./f mice was attenuated in the Mmp13ΔPrrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell–derived MMP13 may regulate osteoclast number and/or activity, bone resorption, and bone mass. And increased production of mesenchymal cell-derived factors may be important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone.

Age-related osteoporosis is caused by a deficit in osteoblasts, the cells that secrete bone matrix. The number of osteoblast progenitors also declines with age associated with increased markers of cell senescence. The forkhead box O (FoxO) transcription factors attenuate Wnt/β-catenin signaling and the proliferation of osteoprogenitors, thereby decreasing bone formation. The NAD+-dependent Sirtuin1 (Sirt1) deacetylates FoxOs and β-catenin in osteoblast progenitors and, thereby, increases bone mass. However, it remains unknown whether the Sirt1/FoxO/β-catenin pathway is dysregulated with age in osteoblast progenitors. We found decreased levels of NAD+ in osteoblast progenitor cultures from old mice, associated with increased acetylation of FoxO1 and markers of cell senescence. The NAD+ precursor nicotinamide riboside (NR) abrogated FoxO1 and β-catenin acetylation and several marker of cellular senescence, and increased the osteoblastogenic capacity of cells from old mice. Consistent with these effects, NR administration to C57BL/6 mice counteracted the loss of bone mass with aging. Attenuation of NAD+ levels in osteoprogenitor cultures from young mice inhibited osteoblastogenesis in a FoxO-dependent manner. In addition, mice with decreased NAD+ in cells of the osteoblast lineage lost bone mass at a young age. Together, these findings suggest that the decrease in bone formation with old age is due, at least in part, to a decrease in NAD+ and dysregulated Sirt1/FoxO/β-catenin pathway in osteoblast progenitors. NAD+ repletion, therefore, represents a rational therapeutic approach to skeletal involution.

The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal cells, but functional evidence for the mediators of these effects has only recently begun to emerge. We report that the matrix metalloproteinase 13 (MMP13) is the highest up-regulated gene in mesenchymal cells from mice lacking the estrogen receptor alpha (ERa). In sham-operated female mice with conditional Mmp13 deletion in Prrx1 expressing cells (Mmp13DPrrx1), the femur and tibia length was lower as compared to control littermates (Mmp13f/f). Additionally, in the sham-operated female Mmp13DPrrx1 mice cortical thickness and trabecular bone volume in the femur and tibia were higher and osteoclast number at the endocortical surfaces was lower, whereas bone formation rate was unaffected. Notably, the decrease of cortical thickness caused by ovariectomy (OVX) in the femur and tibia of Mmp13f/f mice was attenuated in the Mmp13DPrrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell–derived MMP13 regulates osteoclast number, bone resorption, and bone mass. And increased production of mesenchymal cell-derived factors are important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone.

Oxidized phospholipids containing phosphocholine (OxPL) are pro-inflammatory lipid peroxidation products that bind to scavenger receptors (SRs), such as Scarb1, and toll-like receptors (TLRs). Excessive OxPL, as found in oxidized low-density lipoprotein (OxLDL), overwhelm these defense mechanisms and become pathogenic in atherosclerosis, nonalcoholic steatohepatitis (NASH), and osteoporosis. We previously reported that the innate IgM natural antibody E06 binds to OxPL and neutralizes their deleterious effects; expression of the single-chain (scFv) form of the antigen-binding domain of E06 (E06-scFv) as a transgene increases trabecular bone in male mice. We show herein that E06-scFv increases trabecular and cortical bone in female and male mice by increasing bone formation and decreasing osteoblast apoptosis in vivo. Homozygous E06-scFv mice have higher bone mass than hemizygous, showing a dose effect of the transgene. To investigate how OxPL restrain bone formation under physiologic conditions, we measured the levels of SRs and TLRs that bind OxPL. We found that osteoblastic cells primarily express Scarb1. Moreover, OxLDL-induced apoptosis and reduced differentiation were prevented in bone marrow-derived or calvaria-derived osteoblasts from Scarb1 knockout mice. Because Scarb1-deficient mice are reported to have high bone mass, our results suggest that E06 may promote bone anabolism in healthy young mice, at least in part, by neutralizing OxPL, which in turn promote Scarb1-mediated apoptosis of osteoblasts or osteoblast precursors. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Small molecules that selectively kill senescent cells (SCs), termed senolytics, have the potential to prevent and treat various age-related diseases and extend healthspan. The use of Bcl-xl inhibitors as senolytics is largely limited by their on-target and dose-limiting platelet toxicity. Here, we report the use of proteolysis-targeting chimera (PROTAC) technology to reduce the platelet toxicity of navitoclax (also known as ABT263), a Bcl-2 and Bcl-xl dual inhibitor, by converting it into PZ15227 (PZ), a Bcl-xl PROTAC, which targets Bcl-xl to the cereblon (CRBN) E3 ligase for degradation. Compared to ABT263, PZ is less toxic to platelets, but equally or slightly more potent against SCs because CRBN is poorly expressed in platelets. PZ effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvement, Bcl-xl PROTACs have the potential to become safer and more potent senolytic agents than Bcl-xl inhibitors., Senolytics have the potential to extend healthspan by selectively killing senescent cells (SCs), but senolytics that target Bcl-xl may cause platelet toxicity. Here, the authors generated a Bcl-xl proteolysis-targeting chimera (PROTAC) senolytic, which effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia.

CXCL12 is abundantly expressed in reticular cells associated with the perivascular niches of the bone marrow (BM) and is indispensable for B lymphopoiesis. Cxcl12 promotes osteoclastogenesis and has been implicated in pathologic bone resorption. We had shown earlier that estrogen receptor α deletion in osteoprogenitors and estrogen deficiency in mice increase Cxcl12 mRNA and protein levels in the BM plasma, respectively. We have now generated female and male mice with conditional deletion of a Cxcl12 allele in Prrx1 targeted cells (Cxcl12∆Prrx1 ) and show herein that they have a 90% decrease in B lymphocytes but increased erythrocytes and adipocytes in the marrow. Ovariectomy increased the expression of Cxcl12 and B-cell number in the Cxcl12f/f control mice, but these effects were abrogated in the Cxcl12∆Prrx1 mice. Cortical bone mass was not affected in Cxcl12∆Prrx1 mice. Albeit, the cortical bone loss caused by ovariectomy was greatly attenuated. Most unexpectedly, the rate of bone turnover in sex steroid-sufficient female or male Cxcl12∆Prrx1 mice was dramatically increased, as evidenced by a more than twofold increase in several osteoblast- and osteoclast-specific mRNAs, as well as increased mineral apposition and bone formation rate and increased osteoclast number in the endosteal surface. The magnitude of the Cxcl12∆Prrx1 -induced changes were much greater than those caused by ovariectomy or orchidectomy in the Cxcl12f/f mice. These results strengthen the evidence that CXCL12 contributes to the loss of cortical bone mass caused by estrogen deficiency. Moreover, they reveal for the first time that in addition to its effects on hematopoiesis, CXCL12 restrains bone turnover-without changing the balance between resorption and formation-by suppressing osteoblastogenesis and the osteoclastogenesis support provided by cells of the osteoblast lineage. © 2020 American Society for Bone and Mineral Research.

The damaging effects of ionizing radiation (IR) on bone mass are well-documented in mice and humans and are most likely due to increased osteoclast number and function. However, the mechanisms leading to inappropriate increases in osteoclastic bone resorption are only partially understood. Here, we show that exposure to multiple fractions of low-doses (10 fractions of 0.4 Gy total body irradiation [TBI]/week, i.e., fractionated exposure) and/or a single exposure to the same total dose of 4 Gy TBI causes a decrease in trabecular, but not cortical, bone mass in young adult male mice. This damaging effect was associated with highly activated bone resorption. Both osteoclast differentiation and maturation increased in cultures of bone marrow-derived macrophages from mice exposed to either fractionated or singular TBI. IR also increased the expression and enzymatic activity of mitochondrial deacetylase Sirtuin-3 (Sirt3)—an essential protein for osteoclast mitochondrial activity and bone resorption in the development of osteoporosis. Osteoclast progenitors lacking Sirt3 exposed to IR exhibited impaired resorptive activity. Taken together, targeting impairment of osteoclast mitochondrial activity could be a novel therapeutic strategy for IR-induced bone loss, and Sirt3 is likely a major mediator of this effect.

Space is a high-stress environment. One major risk factor for the astronauts when they leave the Earth’s magnetic field is exposure to ionizing radiation from galactic cosmic rays (GCR). Several adverse changes occur in mammalian anatomy and physiology in space, including bone loss. In this study, we assessed the effects of simplified GCR exposure on skeletal health in vivo. Three months following exposure to 0.5 Gy total body simulated GCR, blood, bone marrow and tissue were collected from 9 months old male mice. The key findings from our cell and tissue analysis are (1) GCR induced femoral trabecular bone loss in adult mice but had no effect on spinal trabecular bone. (2) GCR increased circulating osteoclast differentiation markers and osteoclast formation but did not alter new bone formation or osteoblast differentiation. (3) Steady-state levels of mitochondrial reactive oxygen species, mitochondrial and non-mitochondrial respiration were increased without any changes in mitochondrial mass in pre-osteoclasts after GCR exposure. (4) Alterations in substrate utilization following GCR exposure in pre-osteoclasts suggested a metabolic rewiring of mitochondria. Taken together, targeting radiation-mediated mitochondrial metabolic reprogramming of osteoclasts could be speculated as a viable therapeutic strategy for space travel induced bone loss.

Hem1 (Hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity and for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass due to defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially due to cytoskeletal disorganization and decreased mitochondrial respiration. Transplantation of bone marrow hematopoietic stem and progenitor cells from wild-type into Hem1 KO mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.

The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal lineage cells, but functional evidence for the precise molecular mechanism(s) and the mediators of these effects has only recently began to emerge. We report that the matrix metalloproteinase 13 (MMP-13) is the highest up-regulated gene in calvaria or bone marrow cells from mice lacking the estrogen receptor (ER) alpha in osteoprogenitors. We, therefore, generated mice with conditional Mmp-13 deletion in Prrx1 expressing cells (Mmp-13ΔPrrx1) and compared the effect of estrogen deficiency on their bone phenotype to that of control littermates (Mmp-13f/f). Femur and tibia length was decreased in sham-operated Mmp-13ΔPrrx1 mice as compared to Mmp-13f/f. Cortical thickness and trabecular bone volume in the femur and tibia were increased and osteoclast number at the endocortical surfaces was decreased in the sham-operated female Mmp-13ΔPrrx1 mice; whereas bone formation rate was unaffected. Ovariectomy (OVX) caused a decrease of cortical thickness in the femur and tibia of Mmp-13f/f control mice. This effect was attenuated in the Mmp-13ΔPrrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell–derived MMP-13 regulates osteoclast number, bone resorption, and bone mass. We have recently reported that the loss of cortical, but not trabecular bone, caused by OVX is also attenuated in Cxcl12ΔPrrx1 mice. Together with the present report, this functional genetic evidence provides proof of principle that increased production of mesenchymal cell-derived factors, such as CXCL12 and MMP-13, are important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone. Therefore, the mechanisms responsible for the protective effect of estrogens on these two major bone compartments are different.

In aging mice, osteoclast number increases in cortical bone but declines in trabecular bone, suggesting that different mechanisms underlie age-associated bone loss in these 2 compartments. Osteocytes produce the osteoclastogenic cytokine RANKL, encoded by Tnfsf11. Tnfsf11 mRNA increases in cortical bone of aged mice, suggesting a mechanism underlying the bone loss. To address this possibility, we aged mice lacking RANKL in osteocytes. Whereas control mice lost cortical bone between 8 and 24 months of age, mice lacking RANKL in osteocytes gained cortical bone during this period. Mice of both genotypes lost trabecular bone with age. Osteoclasts increased with age in cortical bone of control mice but not in RANKL conditional knockout mice. Induction of cellular senescence increased RANKL production in murine and human cell culture models, suggesting an explanation for elevated RANKL levels with age. Overexpression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Finally, elimination of senescent cells from aged mice using senolytic compounds reduced Tnfsf11 mRNA in cortical bone. Our results demonstrate the requirement of osteocyte-derived RANKL for age-associated cortical bone loss and suggest that increased Tnfsf11 expression with age results from accumulation of senescent cells in cortical bone., Induction of senescence in osteoblast-like cells stimulates RANKL production, providing a possible explanation for the increased cortical bone resorption that occurs with age.

Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through direct actions on cells of the myeloid lineage. Here, we investigated the molecular mechanism of this effect. We report that 17β-estradiol (E2) decreased osteoclast number by promoting the apoptosis of early osteoclast progenitors, but not mature osteoclasts. This effect was abrogated in cells lacking Bak/Bax—two pro-apoptotic members of the Bcl-2 family of proteins required for mitochondrial apoptotic death. FasL has been previously implicated in the pro-apoptotic actions of E2. However, we show herein that FasL-deficient mice lose bone mass following ovariectomy indistinguishably from FasL-intact controls, indicating that FasL is not a major contributor to the anti-osteoclastogenic actions of estrogens. Instead, using microarray analysis we have elucidated that ERα-mediated estrogen signaling in osteoclast progenitors decreases “oxidative phosphorylation” and the expression of mitochondria complex I genes. Additionally, E2 decreased the activity of complex I and oxygen consumption rate. Similar to E2, the complex I inhibitor Rotenone decreased osteoclastogenesis by promoting osteoclast progenitor apoptosis via Bak/Bax. These findings demonstrate that estrogens decrease osteoclast number by attenuating respiration, and thereby, promoting mitochondrial apoptotic death of early osteoclast progenitors.

Summary Age‐related bone loss in mice results from a decrease in bone formation and an increase in cortical bone resorption. The former is accounted by a decrease in the number of postmitotic osteoblasts which synthesize the bone matrix and is thought to be the consequence of age‐dependent changes in mesenchymal osteoblast progenitors. However, there are no specific markers for these progenitors, and conclusions rely on results from in vitro cultures of mixed cell populations. Moreover, the culprits of such changes remain unknown. Here, we have used Osx1‐Cre;TdRFP mice in which osteoprogenitors express the TdRFP fluorescent protein. We report that the number of TdRFP‐Osx1 cells, freshly isolated from the bone marrow, declines by more than 50% between 6 and 24 months of age in both female and male mice. Moreover, TdRFP‐Osx1 cells from old mice exhibited markers of DNA damage and senescence, such as γH2AX foci, G1 cell cycle arrest, phosphorylation of p53, increased p21CIP 1 levels, as well as increased levels of GATA4 and activation of NF‐κB – two major stimulators of the senescence‐associated secretory phenotype (SASP). Bone marrow stromal cells from old mice also exhibited elevated expression of SASP genes, including several pro‐osteoclastogenic cytokines, and increased capacity to support osteoclast formation. These changes were greatly attenuated by the senolytic drug ABT263. Together, these findings suggest that the decline in bone mass with age is the result of intrinsic defects in osteoprogenitor cells, leading to decreased osteoblast numbers and increased support of osteoclast formation.

In the present study, we investigated whether treadmill training and electroacupuncture (EA) have autonomous or synergistic beneficial effects on deficits caused by neonatal hypoxia‑ischemia in Sprague-Dawley rats. For this purpose, rats subjected to hypoxia-ischemia underwent treadmill training and EA stimulation from 4 to 8 weeks of age. Conventional EA (CEA) and scalp EA (SEA) were delivered by electrical stimulation (2 Hz, 1 mA) at traditional acupoints and at the scalp to the primary motor area, respectively. In the behavioral examination, markedly improved performances in the rotarod test were observed in the rats that underwent treadmill exercise, and in the rats that underwent treadmill exercise and CEA compared to the untreated rats subjected to hypoxia-ischemia. An improvement was also observed in the passive avoidance test in the rats that underwent treadmill training and EA. As shown by western blot analysis, the expression levels of neuronal nuclei (NeuN), 2',3'-cyclic-nucleotide 3'-phosphodiesterase and myelin basic protein (MBP) exhibited a significant decrease in the contralateral subventricular zone (SVZ) of the rats subjected to hypoxia‑ischemia compared to the controls; however, these expression levels increased following treadmill exercise and EA stimulation. As shown by immunohistochemical analyses, the thickness of the corpus callosum and the integrated optical density (IOD) of MBP were significantly increased in the rats subjected to treadmill exercise and EA compared to the untreated rats subjected to hypoxiaa-ischemia. The synergistic effects of treadmill training and EA were also observed in the protein levels and IOD of MBP. A marked increase in the number of bromodeoxyuridine (BrdU)- and BrdU/NeuN-positive cells in the contralateral SVZ was also observed in the rats that underwent treadmill training and EA; the number of BrdU-positive cells was synergistically affected by treadmill training and EA. These results suggest that treadmill training and EA stimulation contribute to the enhancement of behavioral recovery following hypoxia-ischemia via the upregulation of myelin components and neurogenesis. Thus, treatment with EA stimulation, as well as treadmill training offers another treatment option to promote functional recovery in cerebral palsy.

Jiawei Shenqi-wan (JSQW), which comprises Shenqi-wan and two additional medicinal herbs, has been widely used for the treatment of various growth impairments, including cerebral palsy. In the present study, JSQW was administered to hypoxic-ischemic Sprague-Dawley rats that underwent treadmill training from 4–7 weeks of age to examine the beneficial effects of combined JSQW and treadmill therapy. Behavioral examinations were performed and a significant improvement in cylinder test performance was observed in rats treated with treadmill training compared with hypoxic-ischemia rats (P

Myelin loss limits neurological recovery and myelin regeneration and is critical for restoration of function. We recently discovered that global knock-out of the thrombin receptor, also known as Protease Activated Receptor 1 (PAR1), accelerates myelin development. Here we demonstrate that knocking out PAR1 also promotes myelin regeneration. Outcomes in two unique models of myelin injury and repair, that is lysolecithin or cuprizone-mediated demyelination, showed that PAR1 knock-out in male mice improves replenishment of myelinating cells and remyelinated nerve fibers and slows early axon damage. Improvements in myelin regeneration in PAR1 knock-out mice occurred in tandem with a skewing of reactive astrocyte signatures toward a prorepair phenotype. In cell culture, the promyelinating effects of PAR1 loss of function are consistent with possible direct effects on the myelinating potential of oligodendrocyte progenitor cells (OPCs), in addition to OPC-indirect effects involving enhanced astrocyte expression of promyelinating factors, such as BDNF. These findings highlight previously unrecognized roles of PAR1 in myelin regeneration, including integrated actions across the oligodendrocyte and astroglial compartments that are at least partially mechanistically linked to the powerful BDNF-TrkB neurotrophic signaling system. Altogether, findings suggest PAR1 may be a therapeutically tractable target for demyelinating disorders of the CNS.SIGNIFICANCE STATEMENTReplacement of oligodendroglia and myelin regeneration holds tremendous potential to improve function across neurological conditions. Here we demonstrate Protease Activated Receptor 1 (PAR1) is an important regulator of the capacity for myelin regeneration across two experimental murine models of myelin injury. PAR1 is a G-protein-coupled receptor densely expressed in the CNS, however there is limited information regarding its physiological roles in health and disease. Using a combination of PAR1 knock-out mice, oligodendrocyte monocultures and oligodendrocyte-astrocyte cocultures, we demonstrate blocking PAR1 improves myelin production by a mechanism related to effects across glial compartments and linked in part to regulatory actions toward growth factors such as BDNF. These findings set the stage for development of new clinically relevant myelin regeneration strategies.

Both an increase in osteoclast and a decrease in osteoblast numbers contribute to skeletal aging. Markers of cellular senescence, including expression of the cyclin inhibitor p16, increase with aging in several bone cell populations. The elimination of p16‐expressing cells in old mice, using the INK‐ATTAC transgene, increases bone mass indicating that senescent cells contribute to skeletal aging. However, the identity of the senescent cells and the extent to which ablation of p16‐expressing cells may prevent skeletal aging remain unknown. Using mice expressing the p16‐3MR transgene, we examined whether elimination of p16‐expressing cells between 12 and 24 months of age could preserve bone mass; and whether elimination of these cells from 20 to 26 months of age could restore bone mass. The activation of the p16‐3MR transgene by ganciclovir (GCV) greatly diminished p16 levels in the brain, liver, and osteoclast progenitors from the bone marrow. The age‐related increase in osteoclastogenic potential of myeloid cells was also abrogated by GCV. However, GCV did not alter p16 levels in osteocytes—the most abundant cell type in bone—and had no effect on the skeletal aging of p16‐3MR mice. These findings indicate that the p16‐3MR transgene does not eliminate senescent osteocytes but it does eliminate senescent osteoclast progenitors and senescent cells in other tissues, as described previously. Elimination of senescent osteoclast progenitors, in and of itself, has no effect on the age‐related loss of bone mass. Hence, other senescent cell types, such as osteocytes, must be the seminal culprits.

Despite concerted efforts to identify CNS regeneration strategies, an incomplete understanding of how the needed molecular machinery is regulated limits progress. Here we use models of lateral compression and FEJOTA clip contusion-compression spinal cord injury (SCI) to identify the thrombin receptor (Protease Activated Receptor 1 (PAR1)) as an integral facet of this machine with roles in regulating neurite growth through a growth factor- and cholesterol-dependent mechanism. Functional recovery and signs of neural repair, including expression of cholesterol biosynthesis machinery and markers of axonal and synaptic integrity, were all increased after SCI in PAR1 knockout female mice, while PTEN was decreased. Notably, PAR1 differentially regulated HMGCS1, a gene encoding a rate-limiting enzyme in cholesterol production, across the neuronal and astroglial compartments of the intact versus injured spinal cord. Pharmacologic inhibition of cortical neuron PAR1 using vorapaxar in vitro also decreased PTEN and promoted neurite outgrowth in a cholesterol dependent manner, including that driven by suboptimal brain derived neurotrophic factor (BDNF). Pharmacologic inhibition of PAR1 also augmented BDNF-driven HMGCS1 and cholesterol production by murine cortical neurons and by human SH-SY5Y and iPSC-derived neurons. The link between PAR1, cholesterol and BDNF was further highlighted by demonstrating that the deleterious effects of PAR1 over-activation are overcome by supplementing cultures with BDNF, cholesterol or by blocking an inhibitor of adenylate cyclase, Gαi. These findings document PAR1-linked neurotrophic coupling mechanisms that regulate neuronal cholesterol metabolism as an important component of the machinery regulating CNS repair and point to new strategies to enhance neural resiliency after injury.

Old age and sex steroid deficiency are the two most critical factors for the development of osteoporosis. It remains unknown, however, whether the molecular culprits of the two conditions are similar or distinct. We show herein that at 19.5 months of age-a time by which the age-dependent decline of cortical and cancellous bone mass and cortical porosity were fully manifested in C57BL/6J mice-these animals remained functionally estrogen sufficient. Transgenic mice with conditional expression of mitochondria-targeted catalase-a potent H2 O2 inactivating enzyme-in cells of the myeloid lineage (mitoCAT;LysM-Cre mice) were protected from the loss of cortical, but not cancellous, bone caused by gonadectomy in either sex. Consistent with these findings, in vitro studies with ERα-deficient Prx1+ cells and gonadectomized young adult mice showed that in both sexes decreased ERα signaling in Prx1+ cells leads to an increase in SDF1, a.k.a. CXCL12, an osteoclastogenic cytokine whose effects were abrogated in macrophages from mitoCAT;LysM-Cre mice. In contrast to sex steroid deficiency, the adverse effects of aging on either cortical or cancellous bone were unaffected in mitoCAT;LysM-Cre mice. On the other hand, attenuation of H2 O2 generation in cells of the mesenchymal lineage targeted by Prx1-Cre partially prevented the loss of cortical bone caused by old age. Our results suggest the effects of sex steroid deficiency and aging on the murine skeleton are independent and result from distinct mechanisms. In the former, the prevailing mechanism of the cortical bone loss in both sexes is increased osteoclastogenesis caused by estrogen deficiency; this is likely driven, at least in part, by mesenchymal/stromal cell-derived SDF1. Decreased osteoblastogenesis, owing in part to increased H2 O2, combined with increased osteoclastogenesis caused by aging mechanisms independent of estrogen deficiency, are the prevailing mechanisms of the loss of cortical bone with old age. © 2016 American Society for Bone and Mineral Research.

A diet high in fat and sucrose (HFHS), the so-called Western diet promotes metabolic syndrome, a significant co-morbidity for individuals with spinal cord injury (SCI). Here we demonstrate that the spinal cord of mice consuming HFHS expresses reduced insulin-like growth factor 1 (IGF-1) and its receptor and shows impaired tricarboxylic acid cycle function, reductions in PLP and increases in astrogliosis, all prior to SCI. After SCI, Western diet impaired sensorimotor and bladder recovery, increased microgliosis, exacerbated oligodendrocyte loss and reduced axon sprouting. Direct and indirect neural injury mechanisms are suggested since HFHS culture conditions drove parallel injury responses directly and indirectly after culture with conditioned media from HFHS-treated astrocytes. In each case, injury mechanisms included reductions in IGF-1R, SIRT1 and PGC-1α and were prevented by metformin. Results highlight the potential for a Western diet to evoke signs of neural insulin resistance and injury and metformin as a strategy to improve mechanisms of neural neuroprotection and repair.

Metabolic syndrome is a key risk factor and co-morbidity in multiple sclerosis (MS) and other neurological conditions, such that a better understanding of how a high fat diet contributes to oligodendrocyte loss and the capacity for myelin regeneration has the potential to highlight new treatment targets. Results demonstrate that modeling metabolic dysfunction in mice with chronic high fat diet (HFD) consumption promotes loss of oligodendrocyte progenitors across the brain and spinal cord. A number of transcriptomic and metabolomic changes in ER stress, mitochondrial dysfunction, and oxidative stress pathways in HFD-fed mouse spinal cords were also identified. Moreover, deficits in TCA cycle intermediates and mitochondrial respiration were observed in the chronic HFD spinal cord tissue. Oligodendrocytes are known to be particularly vulnerable to oxidative damage, and we observed increased markers of oxidative stress in both the brain and spinal cord of HFD-fed mice. We additionally identified that increased apoptotic cell death signaling is underway in oligodendrocytes from mice chronically fed a HFD. When cultured under high saturated fat conditions, oligodendrocytes decreased both mitochondrial function and differentiation. Overall, our findings show that HFD-related changes in metabolic regulators, decreased mitochondrial function, and oxidative stress contribute to a loss of myelinating cells. These studies identify HFD consumption as a key modifiable lifestyle factor for improved myelin integrity in the adult central nervous system and in addition new tractable metabolic targets for myelin protection and repair strategies.

To evaluate the question of whether protease activated receptor-1 (PAR-1) antagonist is a potential therapeutic target in multiple sclerosis, we treated experimental autoimmune encephalomyelitis (EAE) mice with two PAR-1 antagonists, KC-A0590 and SCH-530348. Treatment with both antagonists resulted in a significant decrease in the clinical characteristics of EAE mice by suppressing demyelination and infiltration of inflammatory cells in the spinal cord and brain, as well as a significantly reducing the increased thrombin and tumor necrosis factor-α. Profound leakage of dextran was observed in the brain of EAE mice. However, treatment with PAR-1 antagonists resulted in the stabilization of vascular endothelial cells and reduced blood-brain barrier breakdown with suppression of inflammatory response. Treatment with PAR-1 antagonists also resulted in down-regulated expression of matrix metalloproteinase-9 and preserved expression of occludin and zonula occludens (ZO)-1 in the brain and their significant expression was confirmed in neurons, astrocytes, and vascular endothelial cells. Finally, endothelial cells and primary cultured astrocytes were treated with PAR-1 antagonists; both antagonists suppressed thrombin-induced breakdown of ZO-1 in endothelial cells and secretion of matrix metalloproteinase-9 in astrocytes. Collectively, our results suggest that PAR-1 antagonist is effective in attenuation of the clinical symptoms of EAE mice by stabilizing the blood-brain barrier and may have therapeutic potential for treatment of multiple sclerosis.

In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of the androgen receptor (AR) in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via estrogen receptor alpha (ERα) signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (AR(f/y);Prx1-Cre or ERα(f/f);Osx1-Cre) or myeloid cell lineage (AR(f/y);LysM-Cre or ERα(f/f);LysM-Cre) and their descendants. Male AR(f/y);Prx1-Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, AR(f/y);LysM-Cre, ERα(f/f);Osx1-Cre, or ERα(f/f);LysM-Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unexpectedly, adult males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls after ORX. Recapitulation of the effects of ORX by AR deletion only in the AR(f/y);Prx1-Cre mice indicates that the effects of androgens on cancellous bone result from AR signaling in osteoblasts-not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s).

Thread embedding acupuncture (TEA) is an acupuncture treatment applied to many diseases in Korean medical clinics because of its therapeutic effects by continuous stimulation to tissues. It has recently been used to enhance facial skin appearance and antiaging, but data from evidence-based medicine are limited. To investigate whether TEA therapy can inhibit skin photoaging by ultraviolet B (UVB) irradiation, we performed analyses for histology, histopathology, in situ zymography and western blot analysis in HR-1 hairless mice. TEA treatment resulted in decreased wrinkle formation and skin thickness (Epidermis;P=0.001versus UV) in UVB irradiated mice and also inhibited degradation of collagen fibers (P=0.010versus normal) by inhibiting proteolytic activity of gelatinase matrix-metalloproteinase-9 (MMP-9). Western blot data showed that activation of c-Jun N-terminal kinase (JNK) induced by UVB (P=0.002versus normal group) was significantly inhibited by TEA treatment (P=0.005versus UV) with subsequent alleviation of MMP-9 activation (P=0.048versus UV). These results suggest that TEA treatment can have anti-photoaging effects on UVB-induced skin damage by maintenance of collagen density through regulation of expression of MMP-9 and related JNK signaling. Therefore, TEA therapy may have potential roles as an alternative treatment for protection against skin damage from aging.

Decreased cortical thickness and increased cortical porosity are the key anatomic changes responsible for osteoporotic fractures in elderly women and men. The cellular basis of these changes is unbalanced endosteal and intracortical osteonal remodeling by the osteoclasts and osteoblasts that comprise the basic multicellular units (BMUs). Like humans, mice lose cortical bone with age, but unlike humans, this loss occurs in the face of sex steroid sufficiency. Mice are therefore an ideal model to dissect age-specific osteoporotic mechanisms. Nevertheless, lack of evidence for endosteal or intracortical remodeling in mice has raised questions about their translational relevance. We show herein that administration of the antiosteoclastogenic cytokine osteoprotegerin to Swiss Webster mice ablated not only osteoclasts, but also endosteal bone formation, demonstrating the occurrence of BMU-based endosteal remodeling. Femoral cortical thickness decreased in aged male and female C57BL/6J mice, as well as F1 hybrids of C57BL/6J and BALB/cBy mice. This decrease was greater in C57BL/6J mice, indicating a genetic influence. Moreover, endosteal remodeling became unbalanced because of increased osteoclast and decreased osteoblast numbers. The porosity of the femoral cortex increased with age but was much higher in females of both strains. Notably, the increased cortical porosity resulted from de novo intracortical remodeling by osteon-like structures. Age-dependent cortical bone loss was associated with increased osteocyte DNA damage, cellular senescence, the senescence-associated secretory phenotype, and increased levels of RANKL. The demonstration of unbalanced endosteal and intracortical remodeling in old mice validates the relevance of this animal model to involutional osteoporosis in humans.

The aim of this study was to determine the effects and underlying mechanism of aripiprazole (APZ) augmentation for cilostazol (CLS)-treated post-ischemic stroke mice that were exposed to chronic mild stress (CMS). Compared to treatment with either APZ or CLS alone, the combined treatment resulted in a greater reduction in depressive behaviors, including anhedonia, despair-like behaviors, and memory impairments. This treatment also significantly reduced atrophic changes in the striatum, cortex, and midbrain of CMS-treated ischemic mice, and inhibited neuronal cell apoptosis, particularly in the striatum and the dentate gyrus of the hippocampus. Greater proliferation of neuronal progenitor cells was also observed in the ipsilateral striatum of the mice receiving combined treatment compared to mice receiving either drug alone. Phosphorylation of the cyclic adenosine monophosphate response element binding protein (CREB) was increased in the striatum, hippocampus, and midbrain of mice receiving combined treatment compared to treatment with either drug alone, particularly in the neurons of the striatum and hippocampus, and dopaminergic neurons of the midbrain. Our results suggest that APZ may augment the antidepressant effects of CLS via co-regulation of the CREB signaling pathway, resulting in the synergistic enhancement of their neuroprotective effects.

Senescent cells (SnCs) accumulate with age. Genetic or pharmacological clearance of SnCs delays several age-associated disorders and extends the healthspan of both progeroid and wild-type (WT) mice (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). In addition, our previous study showed that transient depletion of SnCs could rejuvenated aged hematopoietic stem cells (HSCs) and muscle stem cells in normally aged mice (Chang J et al. Nat Med, 22:78, 2016). These findings suggest that SnCs play a causative role in aging and certain aging-related pathologies, and that selective clearance of SnCs with a senolytic agent that can selectively kill SnCs can be beneficial in part by rejuvenating aged tissue stem cells. Thus, senolytic drugs are potential novel anti-aging agents. However, SnCs can re-accumulate within a few weeks after depletion in mice, suggesting that long-term and intermittent clearance of SnCs is required to extend healthspan, as shown in previous studies (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). It is not known how long-term and intermittent clearance of SnCs affects tissue stem cells such as HSCs because HSCs are mostly quiescent in order to self-renew and prevent exhaustion. We used p16-3MR transgenic mice to evaluate the effect on HSCs of long-term and intermittent clearance of SnCs by ganciclovir (GCV), which selectively kills SnCs in these mice. We treated WT and p16-3MR mice with GCV (25 mg/kg/day x 5 days/per cycle with a 2-week interval between the cycles by ip injection) or saline (vehicle control) from 12 to 24 months of age. GCV effectively prevented SnC accumulation in p16-3MR mice but had no effect in WT mice. In general, aged p16-3MR mice treated with GCV looked healthier than old vehicle-treated p16-3MR mice and old WT mice treated with vehicle or GCV as expected (Baker DJ et al. Nature, 530: 184, 2016). Importantly, GCV abrogated aging-induced increases in p16 expression, p38MAPK activation, and accumulation of DNA double-strand breaks in HSCs in p16-3MR mice. This abrogation was associated with a significant improvement in HSC clonogenicity, suggesting that long-term and intermittent clearance of SnCs prevents HSC aging in naturally aged mice. This suggestion is confirmed by competitive serial and limited dilution HSC transplantation assays, which revealed that HSCs from GCV-treated old p16-3MR mice were functionally indistinguishable from those of young mice. To elucidate the mechanism by which SnC clearance prevents HSC aging, we evaluated the clonogenicity of Lin−Sca1+c-Kit+ (LSK) cells from young or vehicle- or GCV-treated old p16-3MR mice in co-cultures with bone marrow stromal cells (BMSCs) from these mice. We found that clearance of SnCs not only prevented the decline in HSC clonogenic function with aging but also preserved the hematopoietic supporting function of BMSCs. This preservation was associated with reduced levels of Cdkn2a and Cdkn1a mRNAs and senescence-associated secretory phenotype (SASP) factors (such as Il1β, Tnfα and Ccl5), which can adversely affect HSC self-renewal, and decrease of adipogenic and osteoclastic differentiation. In addition, the levels of the HSC self-renewal promoting factor Spp1 mRNA was higher in BMSCs from GCV-treated old p16-3MR mice than in the cells from vehicle-treated old p16-3MR mice. These findings suggest that long-term and intermittent clearance of SnCs can also inhibit BMSC senescence, which contributes to the prevention of HSC aging. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Chang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Kim:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Almeida:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Campisi:Unity Biotechnology: Equity Ownership, Other: co-founder of Unity Biotechnology which develops small-molecule senolytic drugs. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents.

We investigated whether cilostazol, an activator of cyclic adenosine monophosphate (cAMP)-dependent intracellular signaling, could inhibit ultraviolet B (UVB) irradiation-induced photoaging in HR-1 hairless mice. Cilostazol decreased wrinkle formation and skin thickness in UVB-irradiated mice, as well as increased staining of collagen fibers and inhibition of reactive oxygen species (ROS) formation in the skin. Moreover, the proteolytic activities of gelatinase matrix metalloproteinase (MMP)-9 and collagenase MMP-3 were significantly decreased in UVB-irradiated mice treated with cilostazol. Western blotting showed that UVB-induced activation of p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB was significantly inhibited by cilostazol, whereas the activation of Akt was significantly enhanced by cilostazol. Confirmation of localized protein expression in the skin revealed marked p38 MAPK and NF-κB activation that was mainly detected in the dermis. Marked Akt activation was mainly detected in the epidermis. Our results suggest that cilostazol may have anti-photoaging effects on UVB-induced wrinkle formation by maintaining the extracellular matrix density in the dermis, which occurs via regulation of ROS and related p38 MAPK and NF-κB signaling, and subsequent down-regulation of MMPs. Therefore, cilostazol may protect against photoaging-induced wrinkle formation.

We modeled prolonged cerebral hypoperfusion in mice using bilateral common carotid artery stenosis (BCAS) and electroacupuncture (EA) stimulation was applied at two acupoints, Baihui (GV20) and Dazhui (GV14). In behavioral tests of memory, BCAS produced impairments in spatial and short-term memory in mice that were attenuated by therapeutic EA stimulation. Therapeutic use of EA in BCAS also enhanced oligodendrocyte (OL) differentiation from oligodendrocyte precursor cells (OPCs), in association with white matter improvements in the corpus callosum (CC). In PCR analyses of growth factor gene expression, significant positive changes in 3 genes were observed following EA stimulation in BCAS, and here we highlight alterations in neurotrophin-4/5 (NT4/5). We confirmed EA-mediated positive changes in the expression of NT4/5 and its receptor, tyrosine receptor kinase B (TrkB). Treatment of naïve and BCAS + EA animals with a selective TrkB antagonist, ANA-12, produced losses of myelin and cognitive function that were ameliorated by EA therapy. Moreover, following BCAS we observed an EA-dependent increase in phospho-activated CREB (a downstream mediator of NT4/5-TrkB signaling) in OPCs and OLs of the CC. Our results suggest that EA stimulation promotes the recovery of memory function following white matter injury via a mechanism that promotes oligodendrocyte regeneration and involves NT4/5-TrkB signaling.

We explored the neuroprotective effects of a hexane extract from Uncaria sinensis (HEUS) against glutamate-induced toxicity focusing on its anti-apoptotic mechanism in primary cultured cortical neurons. Pretreatment with HEUS resulted in significantly reduced glutamate-induced toxicity in a dose-dependent manner with a decrease in the release of lactate dehydrogenase. Morphological assay and flow cytometry were performed for determination of the type of cell death; according to the results, treatment with HEUS resulted in a significant reduction of glutamate-induced apoptotic cell death. We examined the anti-apoptotic mechanism of HEUS; treatment with HEUS resulted in markedly decreased expression of death receptor (DR)4, which was induced by glutamate stimulation. In contrast, treatment with HEUS resulted in significantly enhanced levels of expression of glutamate-attenuated XIAP and Bcl-2, as well as marked blockade of glutamate-induced Bid cleavage, which inhibits both extrinsic and intrinsic apoptosis pathways. In addition, pretreatment with HEUS resulted in almost complete blockade of glutamate-induced activation of caspases-8, -9 and -3, as well as cleavage of poly (ADP-ribose) polymerase. These findings suggest that the neuroprotective effects of HEUS against glutamate-induced toxicity occur via inhibition of DR4 and expression of anti-apoptotic proteins XIAP and Bcl-2 resulting in effective abrogation of the activation of caspase cascades and promotion of cell survival.

Osteogenesis imperfecta (OI) is a heterogenous group of genetic disorders of bone fragility. OI type V is an autosomal-dominant disease characterized by calcification of the forearm interosseous membrane, radial head dislocation, a subphyseal metaphyseal radiodense line, and hyperplastic callus formation; the causative mutation involved in this disease has not been discovered yet. Using linkage analysis in a four-generation family and whole-exome sequencing, we identified a heterozygous mutation of c.−14C>T in the 5′-untranslated region of a gene encoding interferon-induced transmembrane protein 5 (IFITM5). It completely cosegregated with the disease in three families and occurred de novo in five simplex individuals. Transfection of wild-type and mutant IFITM5 constructs revealed that the mutation added five amino acids (Met-Ala-Leu-Glu-Pro) to the N terminus of IFITM5. Given that IFITM5 expression and protein localization is restricted to the skeletal tissue and IFITM5 involvement in bone formation, we conclude that this recurrent mutation would have a specific effect on IFITM5 function and thus cause OI type V.

Histone deacetylases (HDACs) deacetylate both histones and nonhistone proteins and play a key role in the regulation of physiologic and aberrant gene expression. Inhibition of HDACs has emerged as a promising therapeutic target for cancer and neurologic diseases. In this study we investigated the osteogenic effect and mechanism of action of MS-275, a class I HDAC inhibitor with preference for HDAC1. Both local and systemic administration of MS-275 stimulated bone regeneration in animal models. MS-275 stimulated mRNA expression and activity of the early osteogenic marker tissue-nonspecific alkaline phosphatase (TNAP) in bone tissue and osteogenic cells. By using a series of TNAP promoter deletion constructs and a DNA affinity precipitation assay, we identified DExH-box helicase Dhx36 as a factor that binds to the MS-275 response element in the TNAP promoter. We also found that Dhx36 binding to the MS-275 response element is crucial for MS-275 induction of TNAP transcription. Dhx36 physically interacted with a subset of HDACs (HDAC1 and -4) whose protein levels were downregulated by MS-275, and forced expression of these HDACs blunted the stimulatory effects of MS-275 by a deacetylase activity-independent mechanism(s). Taken together, the results of our study show that MS-275 induces TNAP transcription by decreasing the interaction of HDAC1/4 with Dhx36, which can at least in part contribute to the bone anabolic effects of MS-275.