Loss of Nmp4 enhances bone gain from sclerostin antibody administration.

Severe osteoporosis is often treated with one of three Food and Drug Administration (FDA)-approved osteoanabolics. These drugs act by (1) parathyroid hormone (PTH) receptor stimulation using analogues to PTH (teriparatide) or PTH-related peptide (abaloparatide) or by (2) monoclonal antibody neutralization of sclerostin, an innate Wnt inhibitor (Scl-mAb, romosozumab-aqqg). The efficacies of both strategies wane over time. The transcription factor Nmp4 (Nuclear Matrix Protein 4) is expressed in all tissues yet mice lacking this gene are healthy and exhibit enhanced PTH-induced bone formation. Conditional deletion of Nmp4 in mesenchymal stem progenitor cells (MSPCs) phenocopies the elevated response to PTH in global Nmp4-/- mice. However, targeted deletion in later osteoblast stages does not replicate this response. In this study we queried whether loss of Nmp4 improves Scl-mAb potency. Experimental cohorts included global Nmp4-/- and Nmp4+/+ littermates and three conditional knockout models. Nmp4-floxed (Nmp4fl/fl) mice were crossed with mice harboring one of three Cre-drivers (i) Prx1Cre+ targeting MSPCs, (ii) BglapCre+ (mature osteocalcin-expressing osteoblasts), and (iii) Dmp1Cre+ (osteocytes). Female mice were treated with Scl-mAb or 0.9 % saline vehicle for 4 or 7 weeks from 10 weeks of age. Skeletal response was assessed using micro-computed tomography, dual-energy X-ray absorptiometry, bone histomorphometry, and serum analysis. Global Nmp4-/- mice exhibited enhanced Scl-mAb-induced increases in trabecular bone in the femur and spine and a heightened increase in whole body areal bone mineral density compared to global Nmp4+/+ controls. This improved Scl-mAb potency was primarily driven by enhanced increases in bone formation. Nmp4fl/fl;PrxCre+ mice showed an exaggerated Scl-mAb-induced increase in femoral bone but not in the spine since Prrx1 is not expressed in vertebra. The Nmp4fl/fl;BglapCre+ and Nmp4fl/fl;Dmp1Cre+ mice did not exhibit an improved Scl-mAb response. We conclude that Nmp4 expression in MSPCs interferes with the bone anabolic response to anti-sclerostin therapy.

NMP4, an Arbiter of Bone Cell Secretory Capacity and Regulator of Skeletal Response to PTH Therapy.

The skeleton is a secretory organ, and the goal of some osteoporosis therapies is to maximize bone matrix output. Nmp4 encodes a novel transcription factor that regulates bone cell secretion as part of its functional repertoire. Loss of Nmp4 enhances bone response to osteoanabolic therapy, in part, by increasing the production and delivery of bone matrix. Nmp4 shares traits with scaling factors, which are transcription factors that influence the expression of hundreds of genes to govern proteome allocation for establishing secretory cell infrastructure and capacity. Nmp4 is expressed in all tissues and while global loss of this gene leads to no overt baseline phenotype, deletion of Nmp4 has broad tissue effects in mice challenged with certain stressors. In addition to an enhanced response to osteoporosis therapies, Nmp4-deficient mice are less sensitive to high fat diet-induced weight gain and insulin resistance, exhibit a reduced disease severity in response to influenza A virus (IAV) infection, and resist the development of some forms of rheumatoid arthritis. In this review, we present the current understanding of the mechanisms underlying Nmp4 regulation of the skeletal response to osteoanabolics, and we discuss how this unique gene contributes to the diverse phenotypes among different tissues and stresses. An emerging theme is that Nmp4 is important for the infrastructure and capacity of secretory cells that are critical for health and disease.

Conditional Loss of Nmp4 in Mesenchymal Stem Progenitor Cells Enhances PTH-Induced Bone Formation.

Activation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis, yet FDA-approved osteoanabolics, eg, parathyroid hormone (PTH), have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice exhibit enhanced PTH-induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues; therefore, to determine which cell type is responsible for driving the beneficial effects of Nmp4 inhibition, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4-floxed (Nmp4fl/fl ) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre+  to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre+  targeting mature osteoblasts, and (iii) Dmp1Cre+  to disable Nmp4 in osteocytes. Virgin female Cre+  and Cre- mice (10 weeks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1-34 at 30 µg/kg or vehicle for 4 weeks or 7 weeks. Skeletal response was assessed using dual-energy X-ray absorptiometry, micro-computed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4fl/fl ;Prx1Cre+  mice virtually phenocopied the global Nmp4-/- skeleton in the femur, ie, a mild baseline phenotype but significantly enhanced PTH-induced increase in femur trabecular bone volume/total volume (BV/TV) compared with their Nmp4fl/fl ;Prx1Cre- controls. This was not observed in the spine, where Prrx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4fl/fl ;BglapCre+ ) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4fl/fl ;Dmp1Cre+ ) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4-/- Prx1-expressing MSPCs drive the improved response to PTH therapy and that this gene has stage-specific effects on osteoanabolism. © 2022 American Society for Bone and Mineral Research (ASBMR).

NMP4 regulates the innate immune response to influenza A virus infection.

Severe influenza A virus infection typically triggers excessive and detrimental lung inflammation with massive cell infiltration and hyper-production of cytokines and chemokines. We identified a novel function for nuclear matrix protein 4 (NMP4), a zinc-finger-containing transcription factor playing roles in bone formation and spermatogenesis, in regulating antiviral immune response and immunopathology. Nmp4-deficient mice are protected from H1N1 influenza infection, losing only 5% body weight compared to a 20% weight loss in wild type mice. While having no effects on viral clearance or CD8/CD4 T cell or humoral responses, deficiency of Nmp4 in either lung structural cells or hematopoietic cells significantly reduces the recruitment of monocytes and neutrophils to the lungs. Consistent with fewer innate cells in the airways, influenza-infected Nmp4-deficient mice have significantly decreased expression of chemokine genes Ccl2, Ccl7 and Cxcl1 as well as pro-inflammatory cytokine genes Il1b and Il6. Furthermore, NMP4 binds to the promoters and/or conserved non-coding sequences of the chemokine genes and regulates their expression in mouse lung epithelial cells and macrophages. Our data suggest that NMP4 functions to promote monocyte- and neutrophil-attracting chemokine expression upon influenza A infection, resulting in exaggerated innate inflammation and lung tissue damage.

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro.

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def ), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.

Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality.

A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion: a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. The expression of matrix genes that contribute to bone material-level mechanical properties was elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.

Megakaryocytes Enhance Mesenchymal Stromal Cells Proliferation and Inhibit Differentiation.

Megakaryocytes (MKs) can induce proliferation of calvarial osteoblasts [Ciovacco et al., 2009], but this same phenomenon has not been reported for bone marrow stromal populations from long bones. Bone marrow contains several types of progenitor cells which can be induced to differentiate into multiple cell types. Herein, we examined mesenchymal stromal cell proliferation and osteoblastic differentiation when rabbit or mouse MK were cultured with i) rabbit bone marrow stromal cells, ii) rabbit dental pulp stromal cells, or iii) mouse bone marrow stromal cells. Our results demonstrated that rabbit and mouse stromal cells co-cultured with rabbit MK or mouse MK, have significant increases in proliferation on day 7 by 52%, 46%, and 24%, respectively, compared to cultures without MK. Conversely, alkaline phosphatase (ALP) activity was lower at various time points in these cells when cultures contain MK. Similarly, calcium deposition observed at day 14 rabbit bone marrow and dental pulp stromal cells and day 21 mouse bone marrow stromal cells was 63%, 69%, and 30% lower respectively, when co-cultured with MK. Gene expression studies reveal transcriptional changes broadly consistent with increased proliferation and decreased differentiation. Transcript levels of c-fos (associated with cell proliferation) trended higher after 3, 7, and 14 days in culture. Also, expression of alkaline phosphatase, osteonectin, osterix, and osteopontin, which are markers for osteoblast differentiation, showed MK-induced decreases in a cell type and time dependent manner. Taken together, these data suggest that MK play a role in stromal cell proliferation and differentiation, from multiple sites/locations in multiple species. This article is protected by copyright. All rights reserved.

Improving Combination Osteoporosis Therapy in a Preclinical Model of Heightened Osteoanabolism.

Combining anticatabolic agents with parathyroid hormone (PTH) to enhance bone mass has yielded mixed results in osteoporosis patients. Toward the goal of enhancing the efficacy of these regimens, we tested their utility in combination with loss of the transcription factor Nmp4 because disabling this gene amplifies PTH-induced increases in trabecular bone in mice by boosting osteoblast secretory activity. We addressed whether combining a sustained anabolic response with an anticatabolic results in superior bone acquisition compared with PTH monotherapy. Additionally, we inquired whether Nmp4 interferes with anticatabolic efficacy. Wild-type and Nmp4-/- mice were ovariectomized at 12 weeks of age, followed by therapy regimens, administered from 16 to 24 weeks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Anabolic therapeutic efficacy generally corresponded with PTH + RAL = PTH + ZOL > PTH + ALN = PTH > vehicle control. Loss of Nmp4 enhanced femoral trabecular bone increases under PTH + RAL and PTH + ZOL. RAL and ZOL promoted bone restoration, but unexpectedly, loss of Nmp4 boosted RAL-induced increases in femoral trabecular bone. The combination of PTH, RAL, and loss of Nmp4 significantly increased bone marrow osteoprogenitor number, but did not affect adipogenesis or osteoclastogenesis. RAL, but not ZOL, increased osteoprogenitors in both genotypes. Nmp4 status did not influence bone serum marker responses to treatments, but Nmp4-/- mice as a group showed elevated levels of the bone formation marker osteocalcin. We conclude that the heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, in part by increasing hyperanabolic osteoprogenitors. Nmp4 provides a promising target pathway for identifying barriers to pharmacologically induced bone formation.

Nuclear Matrix Protein 4 Is a Novel Regulator of Ribosome Biogenesis and Controls the Unfolded Protein Response via Repression of Gadd34 Expression.

The unfolded protein response (UPR) maintains protein homeostasis by governing the processing capacity of the endoplasmic reticulum (ER) to manage ER client loads; however, key regulators within the UPR remain to be identified. Activation of the UPR sensor PERK (EIFAK3/PEK) results in the phosphorylation of the α subunit of eIF2 (eIF2α-P), which represses translation initiation and reduces influx of newly synthesized proteins into the overloaded ER. As part of this adaptive response, eIF2α-P also induces a feedback mechanism through enhanced transcriptional and translational expression of Gadd34 (Ppp1r15A),which targets type 1 protein phosphatase for dephosphorylation of eIF2α-P to restore protein synthesis. Here we describe a novel mechanism by which Gadd34 expression is regulated through the activity of the zinc finger transcription factor NMP4 (ZNF384, CIZ). NMP4 functions to suppress bone anabolism, and we suggest that this occurs due to decreased protein synthesis of factors involved in bone formation through NMP4-mediated dampening of Gadd34 and c-Myc expression. Loss of Nmp4 resulted in an increase in c-Myc and Gadd34 expression that facilitated enhanced ribosome biogenesis and global protein synthesis. Importantly, protein synthesis was sustained during pharmacological induction of the UPR through a mechanism suggested to involve GADD34-mediated dephosphorylation of eIF2α-P. Sustained protein synthesis sensitized cells to pharmacological induction of the UPR, and the observed decrease in cell viability was restored upon inhibition of GADD34 activity. We conclude that NMP4 is a key regulator of ribosome biogenesis and the UPR, which together play a central role in determining cell viability during endoplasmic reticulum stress.

Genome-Wide Mapping and Interrogation of the Nmp4 Antianabolic Bone Axis.

PTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4(-/-) mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8(+) T cells. To determine whether the Nmp4(-/-) phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4(-/-) mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling on Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 w) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4(-/-) bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4(-/-) mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8(+) T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4(-/-) MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4(-/-) MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues.

Signaling pathways involved in megakaryocyte-mediated proliferation of osteoblast lineage cells.

Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Lennert et al., 1975; Thiele et al., 1999; Chagraoui et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell-cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb-mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK-to-OB signaling ultimately results in significant increases in the expression of c-fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin-mediated signaling mechanism, activating a novel response axis that de-represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone-related diseases.

Mechanical loading in osteocytes induces formation of a Src/Pyk2/MBD2 complex that suppresses anabolic gene expression.

Mechanical stimulation of the skeleton promotes bone gain and suppresses bone loss, ultimately resulting in improved bone strength and fracture resistance. The molecular mechanisms directing anabolic and/or anti-catabolic actions on the skeleton during loading are not fully understood. Identifying molecular mechanisms of mechanotransduction (MTD) signaling cascades could identify new therapeutic targets. Most research into MTD mechanisms is typically focused on understanding the signaling pathways that stimulate new bone formation in response to load. However, we investigated the structural, signaling and transcriptional molecules that suppress the stimulatory effects of loading. The high bone mass phenotype of mice with global deletion of either Pyk2 or Src suggests a role for these tyrosine kinases in repression of bone formation. We used fluid shear stress as a MTD stimulus to identify a novel Pyk2/Src-mediated MTD pathway that represses mechanically-induced bone formation. Our results suggest Pyk2 and Src function as molecular switches that inhibit MTD in our mechanically stimulated osteocyte culture experiments. Once activated by oscillatory fluid shear stress (OFSS), Pyk2 and Src translocate to and accumulate in the nucleus, where they associate with a protein involved in DNA methylation and the interpretation of DNA methylation patterns -methyl-CpG-binding domain protein 2 (MBD2). OFSS-induced Cox-2 and osteopontin expression was enhanced in Pyk2 KO osteoblasts, while inhibition of Src enhanced osteocalcin expression in response to OFSS. We found that Src kinase activity increased in the nucleus of osteocytes in response to OFSS and an interaction activated between Src (Y418) and Pyk2 (Y402) increased in response to OFSS. Thus, as a mechanism to prevent an over-reaction to physical stimulation, mechanical loading may induce the formation of a Src/Pyk2/MBD2 complex in the nucleus that functions to suppress anabolic gene expression.

Functional impairment of bone formation in the pathogenesis of osteoporosis: the bone marrow regenerative competence.

The skeleton is a high-renewal organ that undergoes ongoing cycles of remodeling. The regenerative bone formation arm ultimately declines in the aging, postmenopausal skeleton, but current therapies do not adequately address this deficit. Bone marrow is the primary source of the skeletal anabolic response and the mesenchymal stem cells (MSCs), which give rise to bone matrix-producing osteoblasts. The identity of these stem cells is emerging, but it now appears that the term 'MSC' has often been misapplied to the bone marrow stromal cell (BMSC), a progeny of the MSC. Nevertheless, the changes in BMSC phenotype associated with age and estrogen depletion likely contribute to the attenuated regenerative competence of the marrow and may reflect alterations in MSC phenotype. Here we summarize current concepts in bone marrow MSC identity, and within this context, review recent observations on changes in bone marrow population dynamics associated with aging and menopause.

Nmp4/CIZ suppresses the parathyroid hormone anabolic window by restricting mesenchymal stem cell and osteoprogenitor frequency.

Parathyroid hormone (PTH) anabolic osteoporosis therapy is intrinsically limited by unknown mechanisms. We previously showed that disabling the transcription factor Nmp4/CIZ in mice expanded this anabolic window while modestly elevating bone resorption. This enhanced bone formation requires a lag period to materialize. Wild-type (WT) and Nmp4-knockout (KO) mice exhibited equivalent PTH-induced increases in bone at 2 weeks of treatment, but by 7 weeks, the null mice showed more new bone. At 3-week treatment, serum osteocalcin, a bone formation marker, peaked in WT mice, but continued to increase in null mice. To determine if 3 weeks is the time when the addition of new bone diverges and to investigate its cellular basis, we treated 10-week-old null and WT animals with human PTH (1-34) (30 µg/kg/day) or vehicle before analyzing femoral trabecular architecture and bone marrow (BM) and peripheral blood phenotypic cell profiles. PTH-treated Nmp4-KO mice gained over 2-fold more femoral trabecular bone than WT by 3 weeks. There was no difference between genotypes in BM cellularity or profiles of several blood elements. However, the KO mice exhibited a significant elevation in CFU-F cells, CFU-F(Alk)(Phos+) cells (osteoprogenitors), and a higher percentage of CFU-F(Alk)(Phos+) cells/CFU-F cells consistent with an increase in CD45-/CD146+/CD105+/nestin+ mesenchymal stem cell frequency. Null BM exhibited a 2-fold enhancement in CD8+ T cells known to support osteoprogenitor differentiation and a 1.6-fold increase in CFU-GM colonies (osteoclast progenitors). We propose that Nmp4/CIZ limits the PTH anabolic window by restricting the number of BM stem, progenitor, and blood cells that support anabolic bone remodeling.

Nmp4/CIZ closes the parathyroid hormone anabolic window.

Chronic degenerative diseases are increasing with the aging U.S. population. One consequence of this phenomenon is the need for long-term osteoporosis therapies. Parathyroid hormone (PTH), the only FDA-approved treatment that adds bone to the aged skeleton, loses its potency within two years of initial treatment but the mechanism regulating its limited "anabolic window" is unknown. We have discovered that disabling the nucleocytoplasmic shuttling transcription factor nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) in mice extends the PTH bone-forming capacity. Nmp4 was discovered during our search for nuclear matrix transcription factors that couple this hormone's impact on osteoblast cytoskeletal and nuclear organization with its anabolic capacity. CIZ was independently discovered as a protein that associates with the focal adhesion-associated mechanosensor p130Cas. The Nmp4/CIZ-knockout (KO) skeletal phenotype exhibits a modestly enhanced bone mineral density but manifests an exaggerated response to both PTH and to BMP2 and is resistant to disuse-induced bone loss. The cellular basis of the global Nmp4/CIZ-KO skeletal phenotype remains to be elucidated but may involve an expansion of the bone marrow osteoprogenitor population along with modestly enhanced osteoblast and osteoclast activities supporting anabolic bone turnover. As a shuttling Cys(2)His(2) zinc finger protein, Nmp4/CIZ acts as a repressive transcription factor perhaps associated with epigenetic remodeling complexes, but the functional significance of its interaction with p130Cas is not known. Despite numerous remaining questions, Nmp4/CIZ provides insights into how the anabolic window is regulated, and itself may provide an adjuvant therapy target for the treatment of osteoporosis by extending PTH anabolic efficacy.

Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT).

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism.

Anabolic and catabolic regimens of human parathyroid hormone 1-34 elicit bone- and envelope-specific attenuation of skeletal effects in Sost-deficient mice.

PTH is a potent calcium-regulating factor that has skeletal anabolic effects when administered intermittently or catabolic effects when maintained at consistently high levels. Bone cells express PTH receptors, but the cellular responses to PTH in bone are incompletely understood. Wnt signaling has recently been implicated in the osteo-anabolic response to the hormone. Specifically, the Sost gene, a major antagonist of Wnt signaling, is down-regulated by PTH exposure. We investigated this mechanism by treating Sost-deficient mice and their wild-type littermates with anabolic and catabolic regimens of PTH and measuring the skeletal responses. Male Sost(+/+) and Sost(-/-) mice were injected daily with human PTH 1-34 (0, 30, or 90 µg/kg) for 6 wk. Female Sost(+/+) and Sost(-/-) mice were continuously infused with vehicle or high-dose PTH (40 µg/kg · d) for 3 wk. Dual energy x-ray absorptiometry-derived measures of intermittent PTH (iPTH)-induced bone gain were impaired in Sost(-/-) mice. Further probing revealed normal or enhanced iPTH-induced cortical bone formation rates but concomitant increases in cortical porosity among Sost(-/-) mice. Distal femur trabecular bone was highly responsive to iPTH in Sost(-/-) mice. Continuous PTH (cPTH) infusion resulted in equal bone loss in Sost(+/+) and Sost(-/-) mice as measured by dual energy x-ray absorptiometry. However, distal femur trabecular bone, but not lumbar spine trabecular bone, was spared the bone-wasting effects of cPTH in Sost(-/-) mice. These results suggest that changes in Sost expression are not required for iPTH-induced anabolism. iPTH-induced resorption of cortical bone might be overstimulated in Sost-deficient environments. Furthermore, Sost deletion protects some trabecular compartments, but not cortical compartments, from bone loss induced by high-dose PTH infusion.

Nmp4/CIZ suppresses the response of bone to anabolic parathyroid hormone by regulating both osteoblasts and osteoclasts.

How parathyroid hormone (PTH) increases bone mass is unclear, but understanding this phenomenon is significant to the improvement of osteoporosis therapy. Nmp4/CIZ is a nucleocytoplasmic shuttling transcriptional repressor that suppresses PTH-induced osteoblast gene expression and hormone-stimulated gains in murine femoral trabecular bone. To further characterize Nmp4/CIZ suppression of hormone-mediated bone growth, we treated 10-week-old Nmp4-knockout (KO) and wild-type (WT) mice with intermittent human PTH(1-34) at 30 µg/kg daily or vehicle, 7 days/week, for 2, 3, or 7 weeks. Null mice treated with hormone (7 weeks) gained more vertebral and tibial cancellous bone than WT animals, paralleling the exaggerated response in the femur. Interestingly, Nmp4/CIZ suppression of this hormone-stimulated bone formation was not apparent during the first 2 weeks of treatment. Consistent with the null mice enhanced PTH-stimulated addition of trabecular bone, these animals exhibited an augmented hormone-induced increase in serum osteocalcin 3 weeks into treatment. Unexpectedly, the Nmp4-KO mice displayed an osteoclast phenotype. Serum C-terminal telopeptide, a marker for bone resorption, was elevated in the null mice, irrespective of treatment. Nmp4-KO bone marrow cultures produced more osteoclasts, which exhibited elevated resorbing activity, compared to WT cultures. The expression of several genes critical to the development of both osteoblasts and osteoclasts was elevated in Nmp4-KO mice at 2 weeks, but not 3 weeks, of hormone exposure. We propose that Nmp4/CIZ dampens PTH-induced improvement of trabecular bone throughout the skeleton by transiently suppressing hormone-stimulated increases in the expression of proteins key to the required enhanced activity and number of both osteoblasts and osteoclasts.

Blockade of TNFR1 signaling: A role of oscillatory fluid shear stress in osteoblasts.

Fluid shear stress protects cells from TNF-α-induced apoptosis. Oscillatory fluid shear stress (OFSS) is generally perceived as physiologically relevant biophysical signal for bone cells. Here we identify several cellular mechanisms responsible for mediating the protective effects of OFSS against TNF-α-induced apoptosis in vitro. We found that exposure of MC3T3-E1 osteoblast-like cells to as little as 5 min of OFSS suppressed TNF-α-induced activation of caspase-3, cleavage of PARP and phosphorylation of histone. In contrast, H(2)O(2)-induced apoptosis was not inhibited by OFSS suggesting that OFSS might not be protecting cells from TNF-α-induced apoptosis via stimulation of global pro-survival signaling pathways. In support of this speculation, OFSS inhibition of TNF-α-induced apoptosis was unaffected by inhibitors of several pro-survival signaling pathways including pI3-kinase (LY294002), MAPK/ERK kinase (PD98059 or U0126), intracellular Ca2+ release (U73122), NO production (L-NAME), or protein synthesis (cycloheximide) that were applied to cells during exposure to OFSS and during TNF-α treatment. However, TNF-α-induced phosphorylation and degradation of IκBα was blocked by pre-exposure of cells to OFSS suggesting a more specific effect of OFSS on TNF-α signaling. We therefore focused on the mechanism of OFSS regulation of TNF-receptor 1 (TNFR1) signaling and found that OFSS (1) reduced the amount of receptor on the cell surface, (2) prevented the association of ubiquitinated RIP in TNFR1 complexes with TRADD and TRAF2, and (3) reduced TNF-α-induced IL-8 promoter activity in the nucleus. We conclude that the anti-apoptotic effect of OFSS is not mediated by activation of universal pro-survival signaling pathways. Rather, OFSS inhibits TNF-α-induced pro-apoptotic signaling which can be explained by the down-regulation of TNFR1 on the cell surface and blockade of TNFR1 downstream signaling by OFSS.