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.

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.

Histone Deacetylases in Bone Development and Skeletal Disorders.

Histone deacetylases (Hdacs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins. Eleven of the 18 Hdacs encoded by the human and mouse genomes depend on Zn(2+) for enzymatic activity, while the other 7, the sirtuins (Sirts), require NAD2(+). Collectively, Hdacs and Sirts regulate numerous cellular and mitochondrial processes including gene transcription, DNA repair, protein stability, cytoskeletal dynamics, and signaling pathways to affect both development and aging. Of clinical relevance, Hdacs inhibitors are United States Food and Drug Administration-approved cancer therapeutics and are candidate therapies for other common diseases including arthritis, diabetes, epilepsy, heart disease, HIV infection, neurodegeneration, and numerous aging-related disorders. Hdacs and Sirts influence skeletal development, maintenance of mineral density and bone strength by affecting intramembranous and endochondral ossification, as well as bone resorption. With few exceptions, inhibition of Hdac or Sirt activity though either loss-of-function mutations or prolonged chemical inhibition has negative and/or toxic effects on skeletal development and bone mineral density. Specifically, Hdac/Sirt suppression causes abnormalities in physiological development such as craniofacial dimorphisms, short stature, and bone fragility that are associated with several human syndromes or diseases. In contrast, activation of Sirts may protect the skeleton from aging and immobilization-related bone loss. This knowledge may prolong healthspan and prevent adverse events caused by epigenetic therapies that are entering the clinical realm at an unprecedented rate. In this review, we summarize the general properties of Hdacs/Sirts and the research that has revealed their essential functions in bone forming cells (e.g., osteoblasts and chondrocytes) and bone resorbing osteoclasts. Finally, we offer predictions on future research in this area and the utility of this knowledge for orthopedic applications and bone tissue engineering.

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 [...].

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.

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.

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.

Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affect cartilage development.

Ezh2 is a histone methyltransferase that suppresses osteoblast maturation and skeletal development. We evaluated the role of Ezh2 in chondrocyte lineage differentiation and endochondral ossification. Ezh2 was genetically inactivated in the mesenchymal, osteoblastic, and chondrocytic lineages in mice using the Prrx1-Cre, Osx1-Cre, and Col2a1-Cre drivers, respectively. WT and conditional knockout mice were phenotypically assessed by gross morphology, histology, and micro-CT imaging. Ezh2-deficient chondrocytes in micromass culture models were evaluated using RNA-Seq, histologic evaluation, and Western blotting. Aged mice with Ezh2 deficiency were also evaluated for premature development of osteoarthritis using radiographic analysis. Ezh2 deficiency in murine chondrocytes reduced bone density at 4 weeks of age but caused no other gross developmental effects. Knockdown of Ezh2 in chondrocyte micromass cultures resulted in a global reduction in trimethylation of histone 3 lysine 27 (H3K27me3) and altered differentiation in vitro RNA-Seq analysis revealed enrichment of an osteogenic gene expression profile in Ezh2-deficient chondrocytes. Joint development proceeded normally in the absence of Ezh2 in chondrocytes without inducing excessive hypertrophy or premature osteoarthritis in vivo In summary, loss of Ezh2 reduced H3K27me3 levels, increased the expression of osteogenic genes in chondrocytes, and resulted in a transient post-natal bone phenotype. Remarkably, Ezh2 activity is dispensable for normal chondrocyte maturation and endochondral ossification in vivo, even though it appears to have a critical role during early stages of mesenchymal lineage commitment.

Loss of Hdac3 in osteoprogenitors increases bone expression of osteoprotegerin, improving systemic insulin sensitivity.

Type 2 diabetes is an emerging global health epidemic. Foundations for new therapies are arising from understanding interactions between body systems. Bone-derived factors that reduce RANKL (receptor activator of NF-kappa B ligand) signaling in the liver may prevent insulin resistance and the onset of type 2 diabetes. Here we demonstrate that deletion of the epigenetic regulator, Hdac3, in Osx1-expressing osteoprogenitors prevents insulin resistance induced by high fat diet by increasing serum and skeletal gene expression levels of osteoprotegerin (Opg), a natural inhibitor of RANKL signaling. Removal of one Opg allele in mice lacking Hdac3 in Osx1+ osteoprogenitors increases the insulin resistance of the Hdac3-deficient mice on a high fat diet. Thus, Hdac3-depletion in osteoblasts increases expression of Opg, subsequently preserving insulin sensitivity. The Hdac inhibitor vorinostat also increased Opg transcription and histone acetylation of the Opg locus. These results define a new mechanism by which bone regulates systemic insulin sensitivity.

DNA methylation and FoxO3a regulate PHLPP1 expression in chondrocytes.

The protein phosphatase Phlpp1 is an essential enzyme for proper chondrocyte function. Altered Phlpp1 levels are associated with cancer and degenerative diseases such as osteoarthritis. While much is known about the post-transcriptional mechanisms controlling Phlpp1 levels, transcriptional regulation of the Phlpp1 gene locus is underexplored. We previously showed that CpG methylation of the PHLPP1 promoter is lower in osteoarthritic cartilage than in normal cartilage, and indirectly correlates with gene expression. Here we further defined the effects of DNA methylation on PHLPP1 promoter activity in chondrocytes. We cloned a 1791 bp fragment of the PHLPP1 promoter (-1589:+202) and found that the first 500 bp were required for maximal promoter activity. General methylation of CpG sites within this fragment significantly blunts transcriptional activity, whereas site-specific methyltransferases HhaI or HpaII decrease transcriptional activation by approximately 50%. We located putative FoxO consensus sites within the PHLPP1 promoter region. Inhibition of DNA methylation by incorporation of 5-azacytidine increases Phlpp1 mRNA levels, but FoxO inhibition abolishes this induction. To determine which FoxO transcription factor mediates Phlpp1 expression, we performed overexpression and siRNA-mediated knock down experiments. Overexpression of FoxO3a, but not FoxO1, increases Phlpp1 levels. Likewise, siRNAs targeting FoxO3a, but not FoxO1, diminished Phlpp1 levels. Last, FoxO inhibition increases glycosaminoglycan staining of cultured chondrocytes and leads to concomitant increases in FGF18 and HAS2 expression. Together, these data demonstrate that CpG methylation and FoxO3a regulate PHLPP1 expression.

Histone deacetylase 3 suppresses Erk phosphorylation and matrix metalloproteinase (Mmp)-13 activity in chondrocytes.

Histone deacetylase (Hdac3) inhibitors are emerging therapies for many diseases including cancers and neurological disorders; however, these drugs are teratogens to the developing skeleton. Hdac3 is essential for proper endochondral ossification as its deletion in chondrocytes increases cytokine signaling and the expression of matrix remodeling enzymes. Here we explored the mechanism by which Hdac3 controls matrix metalloproteinase (Mmp)-13 expression in chondrocytes. In Hdac3-depleted chondrocytes, extracellular signal-regulated kinase (Erk)1/2 as well as its downstream substrate, Runx2, were hyperphosphorylated as a result of decreased expression and activity of the Erk1/2 specific phosphatase, Dusp6. Erk1/2 kinase inhibitors and Dusp6 adenoviruses reduced Mmp13 expression and partially rescued matrix production in Hdac3-deficient chondrocytes. Postnatal chondrocyte-specific deletion of Hdac3 with an inducible Col2a1-Cre caused premature production of pErk1/2 and Mmp13 in the growth plate. Thus, Hdac3 controls the temporal and spatial expression of tissue-remodeling genes in chondrocytes to ensure proper endochondral ossification during development.

Histone deacetylase 7 (Hdac7) suppresses chondrocyte proliferation and ß-catenin activity during endochondral ossification.

Histone deacetylases (Hdacs) regulate endochondral ossification by suppressing gene transcription and modulating cellular responses to growth factors and cytokines. We previously showed that Hdac7 suppresses Runx2 activity and osteoblast differentiation. In this study, we examined the role of Hdac7 in postnatal chondrocytes. Hdac7 was highly expressed in proliferating cells within the growth plate. Postnatal tissue-specific ablation of Hdac7 with a tamoxifen-inducible collagen type 2a1-driven Cre recombinase increased proliferation and ß-catenin levels in growth plate chondrocytes and expanded the proliferative zone. Similar results were obtained in primary chondrocyte cultures where Hdac7 was deleted with adenoviral-Cre. Hdac7 bound ß-catenin in proliferating chondrocytes, but stimulation of chondrocyte maturation promoted the translocation of Hdac7 to the cytoplasm where it was degraded by the proteasome. As a result, ß-catenin levels and transcription activity increased in the nucleus. These data demonstrate that Hdac7 suppresses proliferation and ß-catenin activity in chondrocytes. Reducing Hdac7 levels in early chondrocytes may promote the expansion and regeneration of cartilage tissues.

Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation.

Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1(-/-) chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2.

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Histone deacetylase 3 suppression increases PH domain and leucine-rich repeat phosphatase (Phlpp)1 expression in chondrocytes to suppress Akt signaling and matrix secretion.

HDACs epigenetically regulate cellular processes by modifying chromatin and influencing gene expression. We previously reported that conditional deletion of Hdac3 in osteo-chondroprogenitor cells with Osx1-Cre caused severe osteopenia due to abnormal maturation of osteoblasts. The mice were also smaller. To address the abnormal longitudinal growth in these animals, the role of Hdac3 in chondrocyte differentiation was evaluated. We found that Hdac3 is highly expressed in resting and prehypertrophic growth plate chondrocytes, as well as in articular chondrocytes. Hdac3-deficient chondrocytes entered hypertrophy sooner and were smaller than normal chondrocytes. Extracellular matrix production was suppressed as glycosaminoglycan secretion and production of aggrecan, osteopontin, and matrix extracellular phosphoglycoprotein were reduced in Hdac3-deficient chondrocytes. These phenotypes led to the hypothesis that the Akt/mTOR pathway was repressed in these Hdac3-deficient chondrocytes because Akt promotes hypertrophy and matrix production in many tissues. The phosphorylation and activation of Akt, its substrate mTOR, and the mTOR substrate, p70 S6 kinase, were indeed reduced in Hdac3-deficient primary chondrocytes as well as in chondrocytes exposed to HDAC inhibitors. Expression of constitutively active Akt restored phosphorylation of mTOR and p70 S6K and matrix gene expression levels. Reduced phosphorylation of Akt and its substrates in Hdac3-deficient or HDAC inhibitors treated chondrocytes correlated with increased expression of the phosphatase Phlpp1. Hdac3 associated with a Phlpp1 promoter region containing Smad binding elements and was released after TGFß was added to the culture. These data demonstrate that Hdac3 controls chondrocyte hypertrophy and matrix content by repressing Phlpp1 expression and facilitating Akt activity.

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.

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).

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.

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.