Mouse mandibular-derived osteoclast progenitors have differences in intrinsic properties compared with femoral-derived progenitors.

Craniofacial osteoclasts are essential for site-specific processes such as alveolar bone resorption, tooth eruption, and orthodontic tooth movement. Much of the current understanding of osteoclast development and function comes from studies using long bone-derived cells. Minimal investigation has been done to explore skeletal site differences. The overall goal of this study was to determine if mandibular- and femoral-derived osteoclasts represent distinct populations. To test this hypothesis, bone marrow cells were initially analyzed from the mandible and femur of 2-month-old mice. It was shown that mandibular-derived osteoclasts have enhanced size (mm2) compared with femoral-derived osteoclasts. Since bone marrow macrophages are a heterogenous population, we additionally selected for monocytes and demonstrated that mandibular-derived monocytes also form osteoclasts with increased size compared with femoral-derived monocytes. Osteoclast precursor populations from both skeletal sites were analyzed by flow cytometry. A newly described Ly6CHigh+ population as well as the Ly6Cint population was increased in the mandibular-derived cells. The difference in differentiation potential between monocyte cultures suggests that the increase in the Ly6CHigh+ population may explain the enhanced differentiation potential in mandibular-derived cells. Monocyte genes such as Pu.1, C/ebp-a, and Prdm1 are increased in expression in mandibular-derived monocytes compared with femoral-derived monocytes. As expected with enhanced differentiation, osteoclast genes including Nfatc1, Dc-stamp, Ctsk, and Rank are upregulated in mandibular-derived osteoclast precursors. Future studies will determine how changes in the environment of the mandible lead to changes in percentages of osteoclast progenitors and their differentiation potential.

The circadian regulator PER1 promotes cell reprogramming by inhibiting inflammatory signaling from macrophages.

Circadian regulation of gene expression is prevalent and plays critical roles in cell differentiation. However, its roles in the reprogramming of differentiated cells remain largely unknown. Here, we found that one of the master circadian regulators PER1 promoted virus-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to induced neurons (iNs) and induced pluripotent stem cells (iPSCs). Unexpectedly, PER1 achieved this by repressing inflammatory activation of contaminating macrophages in the MEF culture, rather than by directly modulating the reprogrammability of MEFs. More specifically, we found that transduced viruses activated inflammatory genes in macrophages, such as Tnf encoding TNFα, one of the central inflammatory regulators and an autocrine activator of macrophages. TNFα inhibited iN reprogramming, whereas a TNFα inhibitor promoted iN reprogramming, connecting the inflammatory responses to iN reprogramming. In addition, macrophages were induced to proliferate and mature by non-macrophage cells serving as feeders, which also supported up-regulation of TNFα in macrophages without virus transduction. Furthermore, the 2 inflammatory responses were repressed by the circadian regulator PER1 in macrophages, making reprogrammability dependent on time-of-day of virus transduction. Similar results were obtained with iPSC reprogramming, suggesting a wide occurrence of macrophage-mediated inhibition of cell reprogramming. This study uncovers mechanistic links between cell reprogramming, bystander inflammatory macrophages, and circadian rhythms, which are particularly relevant to in vivo reprogramming and organoid formation incorporating immune cells.

Phlpp1 Expression in Osteoblasts Plays a Modest Role in Bone Homeostasis.

Prior work demonstrated that Phlpp1 deficiency alters limb length and bone mass, but the cell types involved and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within bone-forming osteoblasts, we crossed Phlpp1 floxed mice with mice harboring type 1 collagen (Col1a12.3kb)-Cre. Mineralization of bone marrow stromal cell cultures derived from Phlpp1 cKOCol1a1 was unchanged, but levels of inflammatory genes (eg, Ifng, Il6, Ccl8) and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratios were enhanced by either Phlpp1 ablation or chemical inhibition. Micro-computed tomography of the distal femur and L5 vertebral body of 12-week-old mice revealed no alteration in bone volume per total volume, but compromised femoral bone microarchitecture within Phlpp1 cKOCol1a1 conditional knockout females. Bone histomorphometry of the proximal tibia documented no changes in osteoblast or osteoclast number per bone surface but slight reductions in osteoclast surface per bone surface. Overall, our data show that deletion of Phlpp1 in type 1 collagen-expressing cells does not significantly alter attainment of peak bone mass of either males or females, but may enhance inflammatory gene expression and the ratio of RANKL/OPG. Future studies examining the role of Phlpp1 within models of advanced age, inflammation, or osteocytes, as well as functional redundancy with the related Phlpp2 isoform are warranted. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Conditional Loss of MEF2C Expression in Osteoclasts Leads to a Sex-Specific Osteopenic Phenotype.

Myocyte enhancement factor 2C (MEF2C) is a transcription factor studied in the development of skeletal and smooth muscles. Bone resorption studies have exhibited that the reduced expression of MEF2C contributes to osteopetrosis and the dysregulation of pathological bone remodeling. Our current study aims to determine how MEF2C contributes to osteoclast differentiation and to analyze the skeletal phenotype of Mef2c-cKO mice (Cfms-cre; Mef2cfl/fl). qRT-PCR and Western blot demonstrated that Mef2c expression is highest during the early days of osteoclast differentiation. Osteoclast genes, including c-Fos, c-Jun, Dc-stamp, Cathepsin K, and Nfatc1, had a significant reduction in expression, along with a reduction in osteoclast size. Despite reduced CTX activity, female Mef2c cKO mice were osteopenic, with decreased bone formation as determined via a P1NP ELISA, and a reduced number of osteoblasts. There was no difference between male WT and Mef2c-cKO mice. Our results suggest that Mef2c is critical for osteoclastogenesis, and that its dysregulation leads to a sex-specific osteopenic phenotype.

Macrophages and Intervertebral Disc Degeneration.

The intervertebral disc (IVD) aids in motion and acts to absorb energy transmitted to the spine. With little inherent regenerative capacity, degeneration of the intervertebral disc results in intervertebral disc disease, which contributes to low back pain and significant disability in many individuals. Increasing evidence suggests that IVD degeneration is a disease of the whole joint that is associated with significant inflammation. Moreover, studies show elevated macrophage accumulation within the IVD with increasing levels of disease severity; however, we still need to understand the roles, be they causative or consequential, of macrophages during the degenerative process. In this narrative review, we discuss hallmarks of IVD degeneration, showcase evidence of macrophage involvement during disc degeneration, and explore burgeoning research aimed at understanding the molecular pathways regulating macrophage functions during intervertebral disc degeneration.

Macrophages and Bone Remodeling.

Bone remodeling in the adult skeleton facilitates the removal and replacement of damaged and old bone to maintain bone quality. Tight coordination of bone resorption and bone formation during remodeling crucially maintains skeletal mass. Increasing evidence suggests that many cell types beyond osteoclasts and osteoblasts support bone remodeling, including macrophages and other myeloid lineage cells. Herein, we discuss the origin and functions for macrophages in the bone microenvironment, tissue resident macrophages, osteomacs, as well as newly identified osteomorphs that result from osteoclast fission. We also touch on the role of macrophages during inflammatory bone resorption. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Identification of a novel, MSC-induced macrophage subtype via single-cell sequencing: implications for intervertebral disc degeneration therapy.

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-ß signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-ß neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-ß-dependent mechanisms.

Musculoskeletal Development and Skeletal Pathophysiology's.

Musculoskeletal (MSK) disorders are one of the leading causes of disability for people of all ages and impart significant socio-economic burdens on society [...].

GIRK3 deletion facilitates kappa opioid signaling in chondrocytes, delays vascularization and promotes bone lengthening in mice.

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K+ (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2-/-, Girk3-/- and Girk2/3-/- mice. Bones from 12-week-old Girk2-/- mice were normal in length, but femurs and tibiae from Girk3-/- and Girk2/3-/- mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3-/- mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3-/- chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3-/- mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

Nlrp3 Increases the Host's Susceptibility to Tularemia.

Francisella tularensis (F. tularensis) is a Gram-negative, intracellular bacterium and the causative agent of a fatal human disease known as tularemia. The CDC has classified F. tularensis as a Tier 1 Category A select agent based on its ease of aerosolization, low infectious dose, past use as a bioweapon, and the potential to be used as a bioterror agent. Francisella has a unique replication cycle. Upon its uptake, Francisella remains in the phagosomes for a short period and then escapes into the cytosol, where the replication occurs. Francisella is recognized by cytosolic pattern recognition receptors, Absent In Melanoma 2 (Aim2) and Nacht LRR and PYD domains containing Protein 3 (Nlrp3). The recognition of Francisella ligands by Aim2 and Nlrp3 triggers the assembly and activation of the inflammasome. The mechanism of activation of Aim2 is well established; however, how Nlrp3 inflammasome is activated in response to F. tularensis infection is not known. Unlike Aim2, the protective role of Nlrp3 against Francisella infection is not fully established. This study investigated the role of Nlrp3 and the potential mechanisms through which Nlrp3 exerts its detrimental effects on the host in response to F. tularensis infection. The results from in vitro studies demonstrate that Nlrp3 dampens NF-κB and MAPK signaling, and pro-inflammatory cytokine production, which allows replication of F. tularensis in infected macrophages. In vivo, Nlrp3 deficiency results in differential expression of several genes required to induce a protective immune response against respiratory tularemia. Nlrp3-deficient mice mount a stronger innate immune response, clear bacteria efficiently with minimal organ damage, and are more resistant to Francisella infection than their wild-type counterparts. Together, these results demonstrate that Nlrp3 enhances the host's susceptibility to F. tularensis by modulating the protective innate immune responses. Collectively, this study advances our understanding of the detrimental role of Nlrp3 in tularemia pathogenesis.

Myeloid Lineage Ablation of Phlpp1 Regulates M-CSF Signaling and Tempers Bone Resorption in Female Mice.

Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKOLysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKOLysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that Phlpp1-/- cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates-including Akt, ERK1/2, and PKCζ-accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF.

Phlpp1 is induced by estrogen in osteoclasts and its loss in Ctsk-expressing cells does not protect against ovariectomy-induced bone loss.

Prior studies demonstrated that deletion of the protein phosphatase Phlpp1 in Ctsk-Cre expressing cells enhances bone mass, characterized by diminished osteoclast activity and increased coupling to bone formation. Due to non-specific expression of Ctsk-Cre, the definitive mechanism for this observation was unclear. To further define the role of bone resorbing osteoclasts, we performed ovariectomy (Ovx) and Sham surgeries on Phlpp1 cKOCtsk and WT mice. Micro-CT analyses confirmed enhanced bone mass of Phlpp1 cKOCtsk Sham females. In contrast, Ovx induced bone loss in both groups, with no difference between Phlpp1 cKOCtsk and WT mice. Histomorphometry demonstrated that Ovx mice lacked differences in osteoclasts per bone surface, suggesting that estradiol (E2) is required for Phlpp1 deficiency to have an effect. We performed high throughput unbiased transcriptional profiling of Phlpp1 cKOCtsk osteoclasts and identified 290 differentially expressed genes. By cross-referencing these differentially expressed genes with all estrogen response element (ERE) containing genes, we identified IGFBP4 as potential estrogen-dependent target of Phlpp1. E2 induced PHLPP1 expression, but reduced IGFBP4 levels. Moreover, genetic deletion or chemical inhibition of Phlpp1 was correlated with IGFBP4 levels. We then assessed IGFBP4 expression by osteoclasts in vivo within intact 12-week-old females. Modest IGFBP4 immunohistochemical staining of TRAP+ osteoclasts within WT females was observed. In contrast, TRAP+ bone lining cells within intact Phlpp1 cKOCtsk females robustly expressed IGFBP4, but levels were diminished within TRAP+ bone lining cells following Ovx. These results demonstrate that effects of Phlpp1 conditional deficiency are lost following Ovx, potentially due to estrogen-dependent regulation of IGFBP4.

Pleckstrin homology (PH) domain and Leucine Rich Repeat Phosphatase 1 (Phlpp1) Suppresses Parathyroid Hormone Receptor 1 (Pth1r) Expression and Signaling During Bone Growth.

Endochondral ossification is tightly controlled by a coordinated network of signaling cascades including parathyroid hormone (PTH). Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 (Phlpp1) affects endochondral ossification by suppressing chondrocyte proliferation in the growth plate, longitudinal bone growth, and bone mineralization. As such, Phlpp1-/- mice have shorter long bones, thicker growth plates, and proportionally larger growth plate proliferative zones. The goal of this study was to determine how Phlpp1 deficiency affects PTH signaling during bone growth. Transcriptomic analysis revealed greater PTH receptor 1 (Pth1r) expression and enrichment of histone 3 lysine 27 acetylation (H3K27ac) at the Pth1r promoter in Phlpp1-deficient chondrocytes. PTH (1-34) enhanced and PTH (7-34) attenuated cell proliferation, cAMP signaling, cAMP response element-binding protein (CREB) phosphorylation, and cell metabolic activity in Phlpp1-inhibited chondrocytes. To understand the role of Pth1r action in the endochondral phenotypes of Phlpp1-deficient mice, Phlpp1-/- mice were injected with Pth1r ligand PTH (7-34) daily for the first 4 weeks of life. PTH (7-34) reversed the abnormal growth plate and long-bone growth phenotypes of Phlpp1-/- mice but did not rescue deficits in bone mineral density or trabecular number. These results show that elevated Pth1r expression and signaling contributes to increased proliferation in Phlpp1-/- chondrocytes and shorter bones in Phlpp1-deficient mice. Our data reveal a novel molecular relationship between Phlpp1 and Pth1r in chondrocytes during growth plate development and longitudinal bone growth. © 2021 American Society for Bone and Mineral Research (ASBMR).

Pain and Activity Measurements.

Musculoskeletal pain contributes significantly to chronic pain experienced by adults and to health care use. This chapter details several methods to evaluate pain and physical activity in mice that can be applied to preclinical orthopedic models. These methods include the von Frey filament assay that measures mechanical allodynia, open-field activity assays for evaluation of ambulation, and incapacitance measurements to determine static weight bearing.

Serine/threonine phosphatases in osteoclastogenesis and bone resorption.

Maintenance of optimal bone mass is controlled through the concerted functions of several cell types, including bone resorbing osteoclasts. Osteoclasts function to remove calcified tissue during developmental bone modeling, and degrade bone at sites of damage during bone remodeling. Changes to bone homeostasis can arise with alterations in osteoclastogenesis and/or catabolic activity that are not offset by anabolic activity; thus, factors that regulate osteoclastogenesis and bone resorption are of interest to further our understanding of basic bone biology, and as potential targets for therapeutic intervention. Several key cytokines, including RANKL and M-CSF, as well as co-stimulatory factors elicit kinase signaling cascades that promote osteoclastogenesis. These kinase cascades are offset by the action of protein phosphatases, including members of the serine/threonine phosphatase family. Here we review the functions of serine/threonine phosphatases and their control of osteoclast differentiation and function, while highlighting deficiencies in our understanding of this understudied class of proteins within the field.

Hdac3 deletion in myeloid progenitor cells enhances bone healing in females and limits osteoclast fusion via Pmepa1.

Previous studies examining the role of the histone deacetylase Hdac3 within myeloid cells demonstrated that Hdac3 promotes M2 activation and tissue healing in inflammatory conditions. Since myeloid lineage cells are required for proper bone formation and regeneration, in this study we examined the functions of Hdac3 during bone healing. Conditional deletion of Hdac3 within myeloid progenitors accelerates healing of cortical bone defects. Moreover, reduced osteoclast numbers within the defect site are correlated with Hdac3 suppression. Ex vivo osteoclastogenesis assays further demonstrate that Hdac3 deficiency limits osteoclastogenesis, the number of nuclei per cell and bone resorption, suggesting a defect in cell fusion. High throughput RNA sequencing identified the transmembrane protein Pmepa1 as a differentially expressed gene within osteoclast progenitor cells. Knockdown of Pmepa1 partially restores defects in osteoclastogenesis induced by Hdac3 deficiency. These results show that Hdac3 is required for optimal bone healing and osteoclast fusion, potentially via its regulation of Pmepa1 expression.

Hdac3 regulates bone modeling by suppressing osteoclast responsiveness to RANKL.

Hdac3 is a lysine deacetylase that removes acetyl groups from histones and additional proteins. Although Hdac3 functions within mesenchymal lineage skeletal cells are defined, little is known about Hdac3 activities in bone-resorbing osteoclasts. In this study we conditionally deleted Hdac3 within Ctsk-expressing cells and examined the effects on bone modeling and osteoclast differentiation in mice. Hdac3 deficiency reduced femur and tibia periosteal circumference and increased cortical periosteal osteoclast number. Trabecular bone was likewise reduced and was accompanied by increased osteoclast number per trabecular bone surface. We previously showed that Hdac3 deacetylates the p65 subunit of the NF-κB transcriptional complex to decrease DNA-binding and transcriptional activity. Hdac3-deficient osteoclasts demonstrate increased K310 NF-κB acetylation and NF-κB transcriptional activity. Hdac3-deficient osteoclast lineage cells were hyper-responsive to RANKL and showed elevated ex vivo osteoclast number and size and enhanced bone resorption in pit formation assays. Osteoclast-directed Hdac3 deficiency decreased cortical and trabecular bone mass parameters, suggesting that Hdac3 regulates coupling of bone resorption and bone formation. We surveyed a panel of osteoclast-derived coupling factors and found that Hdac3 suppression diminished sphingosine-1-phosphate production. Osteoclast-derived sphingosine-1-phosphate acts in paracrine to promote bone mineralization. Mineralization of WT bone marrow stromal cells cultured with conditioned medium from Hdac3-deficient osteoclasts was markedly reduced. Expression of alkaline phosphatase, type 1a1 collagen, and osteocalcin was also suppressed, but no change in Runx2 expression was observed. Our results demonstrate that Hdac3 controls bone modeling by suppressing osteoclast lineage cell responsiveness to RANKL and coupling to bone formation.

Phlpp1 is associated with human intervertebral disc degeneration and its deficiency promotes healing after needle puncture injury in mice.

Back pain is a leading cause of global disability and is strongly associated with intervertebral disc (IVD) degeneration (IDD). Hallmarks of IDD include progressive cell loss and matrix degradation. The Akt signaling pathway regulates cellularity and matrix production in IVDs and its inactivation is known to contribute to a catabolic shift and increased cell loss via apoptosis. The PH domain leucine-rich repeat protein phosphatase (Phlpp1) directly regulates Akt signaling and therefore may play a role in regulating IDD, yet this has not been investigated. The aim of this study was to investigate if Phlpp1 has a role in Akt dysregulation during IDD. In human IVDs, Phlpp1 expression was positively correlated with IDD and the apoptosis marker cleaved Caspase-3, suggesting a key role of Phlpp1 in the progression of IDD. In mice, 3 days after IVD needle puncture injury, Phlpp1 knockout (KO) promoted Akt phosphorylation and cell proliferation, with less apoptosis. At 2 and 8 months after injury, Phlpp1 deficiency also had protective effects on IVD cellularity, matrix production, and collagen structure as measured with histological and immunohistochemical analyses. Specifically, Phlpp1-deletion resulted in enhanced nucleus pulposus matrix production and more chondrocytic cells at 2 months, and increased IVD height, nucleus pulposus cellularity, and extracellular matrix deposition 8 months after injury. In conclusion, Phlpp1 has a role in limiting cell survival and matrix degradation in IDD and research targeting its suppression could identify a potential therapeutic target for IDD.

Deficiency in the phosphatase PHLPP1 suppresses osteoclast-mediated bone resorption and enhances bone formation in mice.

Enhanced osteoclast-mediated bone resorption and diminished formation may promote bone loss. Pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (Phlpp1) regulates protein kinase C (PKC) and other proteins in the control of bone mass. Germline Phlpp1 deficiency reduces bone volume, but the mechanisms remain unknown. Here, we found that conditional Phlpp1 deletion in murine osteoclasts increases their numbers, but also enhances bone mass. Despite elevating osteoclasts, Phlpp1 deficiency did not increase serum markers of bone resorption, but elevated serum markers of bone formation. These results suggest that Phlpp1 suppresses osteoclast formation and production of paracrine factors controlling osteoblast activity. Phlpp1 deficiency elevated osteoclast numbers and size in ex vivo osteoclastogenesis assays, accompanied by enhanced expression of proto-oncogene C-Fms (C-Fms) and hyper-responsiveness to macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Although Phlpp1 deficiency increased TRAP+ cell numbers, it suppressed actin-ring formation and bone resorption in these assays. We observed that Phlpp1 deficiency increases activity of PKCζ, a PKC isoform controlling cell polarity, and that addition of a PKCζ pseudosubstrate restores osteoclastogenesis and bone resorption of Phlpp1-deficient osteoclasts. Moreover, Phlpp1 deficiency increased expression of the bone-coupling factor collagen triple helix repeat-containing 1 (Cthrc1). Conditioned growth medium derived from Phlpp1-deficient osteoclasts enhanced mineralization of ex vivo osteoblast cultures, an effect that was abrogated by Cthrc1 knockdown. In summary, Phlpp1 critically regulates osteoclast numbers, and Phlpp1 deficiency enhances bone mass despite higher osteoclast numbers because it apparently disrupts PKCζ activity, cell polarity, and bone resorption and increases secretion of bone-forming Cthrc1.