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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.

A bidirectional and complex relationship exists between bone and glycemia. Persons with type 2 diabetes (T2D) are at risk for bone loss and fracture, however, heightened osteoanabolism may ameliorate T2D-induced deficits in glycemia as bone-forming osteoblasts contribute to energy metabolism via increased glucose uptake and cellular glycolysis. Mice globally lacking Nuclear Matrix Protein 4 (Nmp4), a transcription factor expressed in all tissues and conserved between humans and rodents, are healthy and exhibit enhanced bone formation in response to anabolic osteoporosis therapies. To test whether loss of Nmp4 similarly impacted bone deficits caused by diet induced obesity, male wild type (WT) and Nmp4(−/−) mice (8wks) were fed either low-fat diet (LFD) or high-fat diet (HFD) for 12wks. Endpoint parameters included bone architecture, structural and estimated tissue level mechanical properties, body weight/composition, glucose-stimulated insulin secretion, glucose tolerance, insulin tolerance and metabolic cage analysis. HFD diminished bone architecture and ultimate force and stiffness equally in both genotypes. Unexpectedly, the Nmp4(−/−) mice exhibited deficits in pancreatic β-cell function and were modestly glucose intolerant under normal diet conditions. Despite the β-cell deficits, the Nmp4(−/−) mice were less sensitive to HFD-induced weight gain, increases in % fat mass, and decreases in glucose tolerance and insulin sensitivity. We conclude that Nmp4 supports pancreatic β-cell function but suppresses peripheral glucose utilization, perhaps contributing to its suppression of induced skeletal anabolism. Selective disruption of Nmp4 in peripheral tissues may provide a strategy for improving both induced osteoanabolism and energy metabolism in comorbid patients.

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.

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.

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.

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.

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.

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.

Parathyroid hormone (PTH) restores bone mass to the osteopenic skeleton, but significant questions remain as to the underlying mechanisms. The receptor for advanced glycation end products (RAGE) is a multiligand receptor of the immunoglobulin superfamily; however, recent studies indicate a role in bone physiology. We investigated the significance of RAGE to hormone-induced increases in bone by treating 10-wk-old female Rage-knockout (KO) and wild-type (WT) mice with human PTH-(1–34) at 30 μg·kg−1·day−1 or vehicle control, 7 days/wk, for 7 wk. PTH produced equivalent relative gains in bone mineral density (BMD) and bone mineral content (BMC) throughout the skeleton in both genotypes. PTH-mediated relative increases in cortical area of the midshaft femur were not compromised in the null mice. However, the hormone-induced gain in femoral cancellous bone was significantly attenuated in Rage-KO mice. The loss of RAGE impaired PTH-mediated increases in femoral cancellous bone volume, connectivity density, and trabecular number but did not impact increases in trabecular thickness or decreases in trabecular spacing. Disabling RAGE reduced femoral expression of bone formation genes, but their relative PTH-responsiveness was not impaired. Neutralizing RAGE did not attenuate vertebral cancellous bone response to hormone. Rage-null mice exhibited an attenuated accrual rate of bone mass, with the exception of the spine, and an enhanced accrual rate of fat mass. We conclude that RAGE is necessary for key aspects of the skeleton's response to anabolic PTH. Specifically, RAGE is required for hormone-mediated improvement of femoral trabecular architecture but not intrinsically necessary for increasing cortical thickness.

The expression of matrix metalloproteinase-13 (MMP-13), involved in bone turnover, is elevated in stretched MC3T3-E1 osteoblast-like cells. Strain-mediated forces impact bone remodeling due in large part to the movement of fluid through the canalicular-lacunar network. The resulting fluid shear stress (FSS) over the surface membranes of bone cells initiates bone remodeling. Although the nuclear events mediating putative FSS-induced changes in osteoblast MMP-13 transcription are unknown, previous studies with bone cells suggest an overlap between osteoblast FSS- and PTH-induced signal response pathways. MMP-13 PTH response is regulated by a 110 bp 5' regulatory region, conserved across the mouse, rat, and human genes, that supports the binding of numerous transcription factors including Runx2, c-fos/c-jun, Ets-1, and nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) a nucleocytoplasmic shuttling trans-acting protein that attenuates PTH-driven transcription. Nmp4/CIZ also binds p130(cas), an adaptor protein implicated in mechanotransduction. Here we sought to determine whether Nmp4/CIZ contributes to FSS-induced changes in MMP-13 transcription. FSS (12 dynes/cm(2), 3-5 h) increased MMP-13 promoter-reporter activity approximately two-fold in MC3T3-E1 osteoblast-like cells attended by a comparable increase in mRNA expression. This was accompanied by a decrease in Nmp4/CIZ binding to its cis-element within the PTH response region, the mutation of which abrogated the MMP-13 response to FSS. Interestingly, FSS enhanced Nmp4/CIZ promoter activity and induced p130(cas) nuclear translocation. We conclude that the PTH regulatory region of MMP-13 also contributes to FSS response and that Nmp4/CIZ plays similar but distinct roles in mediating hormone- and FSS-driven induction of MMP-13 in bone cells.

High mobility group box 1 (HMGB1) is a chromatin protein that acts as an immunomodulatory cytokine upon active release from myeloid cells. HMGB1 is also an alarmin, an endogenous molecule released by dying cells that acts to initiate tissue repair. We have previously reported that osteoclasts and osteoblasts release HMGB1 and release by the latter is regulated by parathyroid hormone (PTH), an agent of bone remodeling. A recent study suggests that HMGB1 acts as a chemotactic agent to osteoclasts and osteoblasts during endochondral ossification. To explore the potential impact of HMGB1 in the bone microenvironment and its mechanism of release by osseous cells, we characterized the effects of recombinant protein (rHMGB1) on multiple murine bone cell preparations that together exhibit the various cell phenotypes present in bone. We also inquired whether apoptotic bone cells release HMGB1. rHMGB1 enhanced the RANKL/OPG steady state mRNA ratio and dramatically augmented the release of tumor necrosis factor-alpha (TNFalpha) and interleukin-6 (IL6) in osteoblastogenic bone marrow stromal cell (BMSC) cultures but not in the calvarial-derived MC3T3-E1 cells. Interestingly, rHMGB1 promoted GSK-3beta phosphorylation in MC3T3-E1 cells but not in BMSCs. Apoptotic bone cells released HMGB1, including MLO-Y4 osteocyte-like cells. MLO-Y4 release of HMGB1 was coincident with caspase-3 cleavage. Furthermore, the anti-apoptotic action of PTH on MC3T3-E1 cells correlated with the observed decrease in HMGB1 release. Our data suggest that apoptotic bone cells release HMGB1, that within the marrow HMGB1 is a bone resorption signal, and that intramembraneous and endochondral osteoblasts exhibit differential responses to this cytokine.

Nmp4/CIZ proteins (nuclear matrix protein 4/cas interacting zinc finger protein) contribute to gene regulation in bone, blood, and testis. In osteoblasts, they govern the magnitude of gene response to osteotropic factors like parathyroid hormone (PTH). Nmp4/CIZ is recurrently involved in acute leukemia and it has been implicated in spermatogenesis. However, these conserved proteins, derived from a single gene, are expressed in numerous tissues indicative of a more generalized housekeeping function in addition to their tissue-specific roles. To address how Nmp4/CIZ expression is governed, we characterized the 5' regulatory region of the mouse Nmp4 gene, located on chromosome 6. Two adjacent promoters P(1) [-2521 nucleotide (nt)/-597 nt] and P(2) (-2521 nt/+1 nt) initiate transcription of alternative first exons (U(1) and U(2)). Both promoters lack TATA and CCAAT boxes but contain initiator sites and CpG islands. Northern analysis revealed expression of both U(1) and U(2) in numerous adult tissues consistent with the constitutive and ubiquitous activity of a housekeeping gene. Sequence analysis identified numerous potential transcription factor-binding sites significant to osteogenesis, hematopoeisis, and gonadal development. The promoters are active in both osteoblast-like cells and in the M12 B-lymphocyte cell line. Low doses of PTH attenuated P(1)/P(2) activity in osteoblast-like cells. The Nmp4/CIZ promoters are autoregulated and deletion analysis identified regions that drive P(1) and P(2) basal activities as well as regions that contain positive and negative regulatory elements affecting transcription. The Nmp4/CIZ promoters comprise a genomic regulatory architecture that supports constitutive expression as well as cell- and tissue-specific regulation.

beta-Catenin plays a dual role in cells: one at cell-cell junctions and one regulating gene transcription together with TCF (T-cell Factor) in the nucleus. Recently, a role for beta-catenin in osteoblast differentiation and gene expression has begun to be elucidated. Herein we investigated the effects of fluid shear stress (FSS) on beta-catenin signaling. FSS is a well-characterized anabolic stimulus for osteoblasts; however, the molecular mechanisms for the effects of this stimulation remain largely unknown. We found that 1 hour of laminar FSS (10 dynes/cm(2)) induced translocation of beta-catenin to the nucleus and activated a TCF-reporter gene. Analysis of upstream signals that may regulate beta-catenin signaling activity revealed two potential mechanisms for increased beta-catenin signaling. First, FSS induced a transient, but significant, increase in the phosphorylation of both glycogen synthase kinase 3beta (GSK-3beta) and Akt. Second, FSS reduced the levels of beta-catenin associated with N-cadherin, suggesting that less sequestration of beta-catenin by cadherins occurs in osteoblasts subjected to FSS. Functional analysts of potential genes regulated by beta-catenin signaling in osteoblasts revealed two novel observations. First, endogenous, nuclear beta-catenin purified from osteoblasts formed a complex with a TCF -binding element in the cyclooxygenase-2 promoter, and, second, overexpression of either a constitutively active beta-catenin molecule or inhibition of GSK-3beta activity increased basal cyclooxygenase-2 levels. Together, these data demonstrate for the first time that FSS modulates the activity of both GSK-3beta and beta-catenin and that these signaling molecules regulate cyclooxygenase-2 expression in osteoblasts.

Parathyroid hormone (PTH) regulation of matrix metalloproteinase-13 ( MMP-13) expression in osteoblasts contributes to normal bone turnover. The PTH response region of the rat MMP-13 gene spans nucleotides (nt) −148 to −38 and supports binding of numerous transcription factors, including Runx2, necessary for osteoblast differentiation, c-Fos/c-Jun, and Ets-1. These trans-acting proteins mediate hormone induction via incompletely defined combinatorial interactions. Within this region, adjacent to the distal Runx2 site, is a homopolymeric(dA:dT) element (−119/−110 nt) that conforms to the consensus site for the novel transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ). This protein regulates bone cell expression of type I collagen and suppresses BMP2-enhanced osteoblast differentiation. The aim of this study was to determine whether Nmp4/CIZ contributes to MMP-13 basal transcription and PTH responsiveness in osteoblasts. Electrophoretic mobility shift analysis confirms Nmp4/CIZ binding within the MMP-13 PTH response region. Mutation of the Nmp4/CIZ element decreases basal activity of an MMP-13 promoter-reporter construct containing the first 1329 nt of the 5′-regulatory region, and overexpression of Nmp4/CIZ protein enhances the activity of the wild-type promoter. The same mutation of the homopolymeric(dA:dT) element enhances the MMP-13 response to PTH and PGE2. Overexpression of Nmp4/CIZ diminishes hormone induction. Mutation of both the homopolymeric(dA:dT) element and the adjacent Runx2 site further augments the PTH response. On the basis of these data and previous studies, we propose that Nmp4/CIZ is a component of a multiprotein assemblage or enhanceosome within the MMP-13 PTH response region and that, within this context, Nmp4/CIZ promotes both basal expression and hormonal synergy.

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.

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. J. Cell. Physiol. 227: 1873–1882, 2012. © 2011 Wiley Periodicals, Inc.

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.

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.

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, H2O2-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. J. Cell. Physiol. 226: 1044–1051, 2011. © 2010 Wiley-Liss, Inc.

Cellular mechanotransduction, the process of converting mechanical signals into biochemical responses within cells, is a critical aspect of bone health. While the effects of mechanical loading on bone are well recognized, elucidating the specific molecular pathways involved in the processing of mechanical signals by bone cells represents a challenge and an opportunity to identify therapeutic strategies to combat bone loss. In this study we have for the first time examined the relationship between the nucleocytoplasmic shuttling transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ) and β-catenin signaling in response to a physiologic mechanical stimulation (oscillatory fluid shear stress, OFSS) in osteoblasts. Using calvaria-derived osteoblasts from Nmp4-deficient and wild-type mice, we found that the normal translocation of β-catenin to the nucleus in osteoblasts that is induced by OFSS is enhanced when Nmp4/CIZ is absent. Furthermore, we found that other aspects of OFSS-induced mechanotransduction generally associated with the β-catenin signaling pathway, including ERK, Akt, and GSK3β activity, as well as expression of the β-catenin-responsive protein cyclin D1 are also enhanced in cells lacking Nmp4/CIZ. Finally, we found that in the absence of Nmp4/CIZ, OFSS-induced cytoskeletal reorganization and the formation of focal adhesions between osteoblasts and the extracellular substrate is qualitatively enhanced, suggesting that Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together these results provide experimental support for the concept that Nmp4/CIZ plays an inhibitory role in the response of bone cells to mechanical stimulation induced by OFSS. J. Cell. Physiol. 223: 435–441, 2010. © 2010 Wiley-Liss, Inc.

An accumulating body of clinical studies demonstrates that once-daily intermittent doses of human parathyroid hormone (1–34) [hPTH (1–34)] reduce the fracture risk in patients with severe osteoporosis (Dempster et al., 2001; Neer et al., 2001). Despite its proven efficacy in reducing fractures, and subsequent FDA approval as an osteoporosis therapy, the cellular and molecular mechanisms of action employed by intermittent PTH remain to be fully elucidated. Upon binding to its receptor (PTHR1), PTH activates a multitude of osteoblast molecular signaling networks, beginning with the cAMP/PKA and the cytosolic calcium/PKC response limbs. Subsequent downstream targets include the activation of the transcription factors CREB and Runx2, downregulation of the osteocyte-derived bone formation inhibitor sclerostin, and initiation of an IGF-1 autocrine/paracrine signaling loop, among others (Feister et al., 2000; Bikle et al., 2002; Swarthout et al., 2002; Hurley et al., 2006; Yakar et al., 2006; Zhang et al., 2006; Leupin et al., 2007; Li et al., 2007; Merciris et al., 2007). The collective end result of these and other PTH-induced networks is the enhanced differentiation of committed osteoblast precursors in the bone marrow, and prolongation of the lifespan of the mature osteoblast via suppressed apoptosis (reviewed in Jilka, 2007; Martin and Seeman, 2007). While much of the investigation of anabolic PTH signal transduction and molecular regulation has been focused on the activation of osteogenic kinases, secretion of osteogenic ligands and ECMs, and expression of osteogenic genes, little is known about the inhibitory mechanisms on bone formation that are also activated by the anabolic signaling events. These mechanisms, though antagonistic to the bone-building effects of the signal, likely serve as an important negative feedback mechanism to prevent hyper-responsiveness to the signal. For example, the anti-apoptotic effect of PTH on osteoblasts requires Runx2-driven transcription of survival genes but PTH also induces the Smurf1-mediated proteasomal degradation of this trans-acting protein (Bellido et al., 2003). This mechanism appears to support a self-limiting response of the hormone’s own anabolic action, though it has not been tested in vivo. Similarly, up-regulation of the CREM gene, the products of which act as transcriptional attenuators, may keep the anabolic response to intermittent PTH in check, by restraining PTH-induced osteoclastogenesis (Liu et al., 2007). Elucidation of the osteo-inhibitory mechanisms that are activated by anabolic PTH could yield an attractive target for osteoporosis therapy, either alone or in conjunction with PTH treatment, by essentially providing a means to suppress the “off signal” that accompanies each anabolic activation cycle. We have potentially identified such a target in a series of experiments conducted over the last 12 years (Alvarez et al., 1997, 1998, 2005; Thunyakitpisal et al., 2001; Shah et al., 2004). Nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) is a trans-acting nucleocytoplasmic shuttling protein that, in osteoblasts, acts as a transcriptional attenuator (Shen et al., 2002; Shah et al., 2004; Morinobu et al., 2005). Nmp4/CIZ suppresses PTH-driven matrix metalloproteinase-13 (MMP-13) gene transcription in MC3T3-E1 osteoblast-like cells (Shah et al., 2004). Similarly BMP2-induced up-regulation of alkaline phosphatase, osteocalcin, and type I collagen are all suppressed in MC3T3-E1 cells over-expressing Nmp4/CIZ (Shen et al., 2002), and BMP2- induced bone formation on adult calvariae in vivo was enhanced by CIZ deficiency (Morinobu et al., 2005). Collectively, our studies indicate that Nmp4/CIZ activation induced by anabolic regimens of PTH might be a mechanism to partially antagonize the osteogenic response of bone tissue to therapeutic doses of PTH. From these data, an obvious question emerges: does the anabolic response to PTH improve when Nmp4/CIZ is disabled/deleted? If so, Nmp4/CIZ might represent an attractive pharmacologic target for inhibition in the search for new osteoporosis therapies. In the present communication, we examine the role of Nmp4/CIZ in normal skeletal homeostasis and in regulating the response of the skeleton to intermittent PTH. We engineered mice with a loss-of-function mutation in Nmp4/CIZ, and measured bone mass, density, structure, and gene expression, at several time points. We further evaluated the ramifications of the null mutation on anabolic PTH signaling by challenging the mice with intermittent PTH for 7 weeks. Nmp4-null mice exhibited a modest gain in skeletal BMD and BMC between 8 and 16 weeks of age as compared to WT animals. Notably, the Nmp4-knockout (KO) animals displayed a significantly enhanced hormone-induced gain in femoral cancellous bone. The present data and our previous in vitro studies support our hypothesis that Nmp4/CIZ is a key component to an inhibitory mechanism of bone formation. As such, it appears to act as a transcriptional attenuator that suppresses osteoid synthesis thus limiting anabolic PTH-mediated gains in cancellous bone.

The death of osteocytes, the terminally differentiated cells of the osteoblast lineage that are embedded in bone and regulate remodeling, is significant to both normal and pathological bone resorption. Apoptotic osteocytes putatively release a clarion signal that enhances the development of the bone-resorbing osteoclasts and targets their migration to the breach in the osteocyte network. This phenomenon is thought to underlie normal repair of bone microdamage and contribute to the etiologies of inflammatory bone loss. The chromatin protein high mobility group box 1 protein (HMGB1) has been identified as an "alarmin" in other tissues. An alarmin is an endogenous molecule released by dead and dying cells that alert the innate immune system to damage and the need for tissue repair. Wang and colleagues presented evidence in a landmark 1999 study showing that released HMGB1 is a lethal mediator of sepsis. Extracellular HMGB1 is a ligand for the toll-like receptors (TLRs) and for the receptor for advanced glycation end products (RAGE) all of which amplify inflammation. Recent studies by our lab and others have shown that HMGB1 is a bone-active cytokine. It is released by apoptotic osteoblasts in vitro, including the MLO-Y4 osteocyte-like cells. Extracellular HMGB1 enhances the expression of RANKL, TNFalpha, and IL6 in osteoblastogenic bone marrow stromal cell cultures, and it is chemotactic to osteoclasts. In this prospectus we will review HMGB1 activity at the immune-bone interface and propose a role for HMGB1 as an osteocyte alarmin and mediator of normal remodeling and inflammatory bone loss.

Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.

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.