Depletion of slow-cycling PDGFRα+ADAM12+ mesenchymal cells promotes antitumor immunity by restricting macrophage efferocytosis.

The capacity to survive and thrive in conditions of limited resources and high inflammation is a major driver of tumor malignancy. Here we identified slow-cycling ADAM12+PDGFRα+ mesenchymal stromal cells (MSCs) induced at the tumor margins in mouse models of melanoma, pancreatic cancer and prostate cancer. Using inducible lineage tracing and transcriptomics, we demonstrated that metabolically altered ADAM12+ MSCs induced pathological angiogenesis and immunosuppression by promoting macrophage efferocytosis and polarization through overexpression of genes such as Gas6, Lgals3 and Csf1. Genetic depletion of ADAM12+ cells restored a functional tumor vasculature, reduced hypoxia and acidosis and normalized CAFs, inducing infiltration of effector T cells and growth inhibition of melanomas and pancreatic neuroendocrine cancer, in a process dependent on TGF-ß. In human cancer, ADAM12 stratifies patients with high levels of hypoxia and innate resistance mechanisms, as well as factors associated with a poor prognosis and drug resistance such as AXL. Altogether, our data show that depletion of tumor-induced slow-cycling PDGFRα+ MSCs through ADAM12 restores antitumor immunity.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Chemical Hybridization of Glucagon and Thyroid Hormone Optimizes Therapeutic Impact for Metabolic Disease.

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.

Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Macrophagic AMPKα1 orchestrates regenerative inflammation induced by glucocorticoids.

Macrophages are key cells after tissue damage since they mediate both acute inflammatory phase and regenerative inflammation by shifting from pro-inflammatory to restorative cells. Glucocorticoids (GCs) are the most potent anti-inflammatory hormone in clinical use, still their actions on macrophages are not fully understood. We show that the metabolic sensor AMP-activated protein kinase (AMPK) is required for GCs to induce restorative macrophages. GC Dexamethasone activates AMPK in macrophages and GC receptor (GR) phosphorylation is decreased in AMPK-deficient macrophages. Loss of AMPK in macrophages abrogates the GC-induced acquisition of their repair phenotype and impairs GC-induced resolution of inflammation in vivo during post-injury muscle regeneration and acute lung injury. Mechanistically, two categories of genes are impacted by GC treatment in macrophages. Firstly, canonical cytokine regulation by GCs is not affected by AMPK loss. Secondly, AMPK-dependent GC-induced genes required for the phenotypic transition of macrophages are co-regulated by the transcription factor FOXO3, an AMPK substrate. Thus, beyond cytokine regulation, GR requires AMPK-FOXO3 for immunomodulatory actions in macrophages, linking their metabolic status to transcriptional control in regenerative inflammation.

Molecular Actions of Glucocorticoids in Cartilage and Bone During Health, Disease, and Steroid Therapy.

Cartilage and bone are severely affected by glucocorticoids (GCs), steroid hormones that are frequently used to treat inflammatory diseases. Major complications associated with long-term steroid therapy include impairment of cartilaginous bone growth and GC-induced osteoporosis. Particularly in arthritis, GC application can increase joint and bone damage. Contrarily, endogenous GC release supports cartilage and bone integrity. In the last decade, substantial progress in the understanding of the molecular mechanisms of GC action has been gained through genome-wide binding studies of the GC receptor. These genomic approaches have revolutionized our understanding of gene regulation by ligand-induced transcription factors in general. Furthermore, specific inactivation of GC signaling and the GC receptor in bone and cartilage cells of rodent models has enabled the cell-specific effects of GCs in normal tissue homeostasis, inflammatory bone diseases, and GC-induced osteoporosis to be dissected. In this review, we summarize the current view of GC action in cartilage and bone. We further discuss future research directions in the context of new concepts for optimized steroid therapies with less detrimental effects on bone.

An SNX10-dependent mechanism downregulates fusion between mature osteoclasts.

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that the fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10- to 100-fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous SNX10-dependent mechanism that downregulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo. This article has an associated First Person interview with the joint first authors of the paper.

Deletion of the chondrocyte glucocorticoid receptor attenuates cartilage degradation through suppression of early synovial activation in murine posttraumatic osteoarthritis.

OBJECTIVE: Disruption of endogenous glucocorticoid signalling in bone cells attenuates osteoarthritis (OA) in aged mice, however, the role of endogenous glucocorticoids in chondrocytes is unknown. Here, we investigated whether deletion of the glucocorticoid receptor, specifically in chondrocytes, also alters OA progression. DESIGN: Knee OA was induced by surgical destabilisation of the medial meniscus (DMM) in male 22-week-old tamoxifen-inducible glucocorticoid receptor knockout (chGRKO) mice and their wild-type (WT) littermates (n = 7-9/group). Mice were harvested 2, 4, 8 and 16 weeks after surgery to examine the spatiotemporal changes in molecular, cellular, and histological characteristics. RESULTS: At all time points following DMM, cartilage damage was significantly attenuated in chGRKO compared to WT mice. Two weeks after DMM, WT mice exhibited increased chondrocyte and synoviocyte hypoxia inducible factor (HIF)-2α expression resulting in extensive synovial activation characterised by synovial thickening and increased interleukin-1 beta expression. At 2 and 4 weeks after DMM, WT mice displayed pronounced chondrocyte senescence and elevated catabolic signalling (reduced Yes-associated protein 1 (YAP1) and increased matrix metalloprotease [MMP]-13 expression). Contrastingly, at 2 weeks after DMM, HIF-2α expression and synovial activation were much less pronounced in chGRKO than in WT mice. Furthermore, chondrocyte YAP1 and MMP-13 expression, as well as chondrocyte senescence were similar in chGRKO-DMM mice and sham-operated controls. CONCLUSION: Endogenous glucocorticoid signalling in chondrocytes promotes synovial activation, chondrocyte senescence and cartilage degradation by upregulation of catabolic signalling through HIF-2α in murine posttraumatic OA. These findings indicate that inhibition of glucocorticoid signalling early after injury may present a promising way to slow osteoarthritic cartilage degeneration.

Estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.

Osteoporosis is an age-dependent serious skeletal disease that leads to great suffering for the patient and high social costs, especially as the global population reaches higher age. Decreasing estrogen levels after menopause result in a substantial bone loss and increased fracture risk, whereas estrogen treatment improves bone mass in women. RSPO3, a secreted protein that modulates WNT signaling, increases trabecular bone mass and strength in the vertebrae of mice, and is associated with trabecular density and risk of distal forearm fractures in humans. The aim of the present study was to determine if RSPO3 is involved in the bone-sparing effect of estrogens. We first observed that estradiol (E2) treatment increases RSPO3 expression in bone of ovariectomized (OVX) mice, supporting a possible role of RSPO3 in the bone-sparing effect of estrogens. As RSPO3 is mainly expressed by osteoblasts in the bone, we used a mouse model devoid of osteoblast-derived RSPO3 (Runx2-creRspo3flox/flox mice) to determine if RSPO3 is required for the bone-sparing effect of E2 in OVX mice. We confirmed that osteoblast-specific RSPO3 inactivation results in a substantial reduction in trabecular bone mass and strength in the vertebrae. However, E2 increased vertebral trabecular bone mass and strength similarly in mice devoid of osteoblast-derived RSPO3 and control mice. Unexpectedly, osteoblast-derived RSPO3 was needed for the full estrogenic response on cortical bone thickness. In conclusion, although osteoblast-derived RSPO3 is a crucial regulator of vertebral trabecular bone, it is required for a full estrogenic effect on cortical, but not trabecular, bone in OVX mice. Thus, estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.NEW & NOTEWORTHY Osteoblast-derived RSPO3 is known to be a crucial regulator of vertebral trabecular bone. Our new findings show that RSPO3 and estrogen regulate trabecular bone independent of each other, but that RSPO3 is necessary for a complete estrogenic effect on cortical bone.

Dexamethasone-mediated inhibition of Notch signalling blocks the interaction of leukaemia and mesenchymal stromal cells.

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by a poor prognosis. Bone marrow mesenchymal stromal cells (BM MSCs) support leukaemic cells in preventing chemotherapy-induced apoptosis. This encouraged us to investigate leukaemia-BM niche-associated signalling and to identify signalling cascades supporting the interaction of leukaemic cells and BM MSC. Our study demonstrated functional differences between MSCs originating from leukaemic (AML MSCs) and healthy donors (HD MSCs). The direct interaction of leukaemic and AML MSCs was indispensable in influencing AML cell proliferation. We further identified an important role for Notch expression and its activation in AML MSCs contributing to the enhanced proliferation of AML cells. Supporting this observation, overexpression of the intracellular Notch domain (Notch ICN) in AML MSCs enhanced AML cells' proliferation. From a therapeutic point of view, dexamethasone treatment impeded Notch signalling in AML MSCs resulting in reduced AML cell proliferation. Concurrent with our data, Notch inhibitors had only a marginal effect on leukaemic cells alone but strongly influenced Notch signalling in AML MSCs and abrogated their cytoprotective function on AML cells. In vivo, dexamethasone treatment impeded Notch signalling in AML MSCs leading to a reduced number of AML MSCs and improved survival of leukaemic mice. In summary, targeting the interaction of leukaemic cells and AML MSCs using dexamethasone or Notch inhibitors might further improve treatment outcomes in AML patients.

Correction: Steppe et al. Bone Mass and Osteoblast Activity Are Sex-Dependent in Mice Lacking the Estrogen Receptor α in Chondrocytes and Osteoblast Progenitor Cells. Int. J. Mol. Sci. 2022, 23, 2902.

The authors would like to make corrections to the reference citations in the original article [...].

Bone Mass and Osteoblast Activity Are Sex-Dependent in Mice Lacking the Estrogen Receptor α in Chondrocytes and Osteoblast Progenitor Cells.

While estrogen receptor alpha (ERα) is known to be important for bone development and homeostasis, its exact function during osteoblast differentiation remains unclear. Conditional deletion of ERα during specific stages of osteoblast differentiation revealed different bone phenotypes, which were also shown to be sex-dependent. Since hypertrophic chondrocytes can transdifferentiate into osteoblasts and substantially contribute to long-bone development, we aimed to investigate the effects of ERα deletion in both osteoblast and chondrocytes on bone development and structure. Therefore, we generated mice in which the ERα gene was inactivated via a Runx2-driven cyclic recombinase (ERαfl/fl; Runx2Cre). We analyzed the bones of 3-month-old ERαfl/fl; Runx2Cre mice by biomechanical testing, micro-computed tomography, and cellular parameters by histology. Male ERαfl/fl; Runx2Cre mice displayed a significantly increased cortical bone mass and flexural rigidity of the femurs compared to age-matched controls with no active Cre-transgene (ERαfl/fl). By contrast, female ERαfl/fl; Runx2Cre mice exhibited significant trabecular bone loss, whereas in cortical bone periosteal and endosteal diameters were reduced. Our results indicate that the ERα in osteoblast progenitors and hypertrophic chondrocytes differentially contributes to bone mass regulation in male and female mice and improves our understanding of ERα signaling in bone cells in vivo.

HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling.

AIMS/HYPOTHESIS: Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. METHODS: Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2AdipoqCre) and performed a large panel of metabolic tests. RESULTS: We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR-HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. CONCLUSIONS/INTERPRETATION: In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. DATA AVAILABILITY: Array data have been submitted to the GEO database at NCBI (GSE148699).

Insulin signaling and reduced glucocorticoid receptor activity attenuate postprandial gene expression in liver.

Hepatic circadian gene transcription is tightly coupled to feeding behavior, which has a profound impact on metabolic disorders associated with diet-induced obesity. Here, we describe a genomics approach to uncover mechanisms controlling hepatic postprandial gene expression. Combined transcriptomic and cistromic analysis identified hundreds of circadian-regulated genes and enhancers controlled by feeding. Postprandial suppression of enhancer activity was associated with reduced glucocorticoid receptor (GR) and Forkhead box O1 (FOXO1) occupancy of chromatin correlating with reduced serum corticosterone levels and increased serum insulin levels. Despite substantial co-occupancy of feeding-regulated enhancers by GR and FOXO1, selective disruption of corticosteroid and/or insulin signaling resulted in dysregulation of specific postprandial regulated gene programs. In combination, these signaling pathways operate a major part of the genes suppressed by feeding. Importantly, the feeding response was disrupted in diet-induced obese animals, which was associated with dysregulation of several corticosteroid- and insulin-regulated genes, providing mechanistic insights to dysregulated circadian gene transcription associated with obesity.

CD163 expression defines specific, IRF8-dependent, immune-modulatory macrophages in the bone marrow.

BACKGROUND: Scavenger receptor CD163 is exclusively expressed on monocytes/macrophages and is widely used as a marker for alternatively activated macrophages. However, the role of CD163 is not yet clear. OBJECTIVES: We sought to examine the function of CD163 in steady-state as well as in sterile and infectious inflammation. METHODS: Expression of CD163 was analyzed under normal and inflammatory conditions in mice. Functional relevance of CD163 was investigated in models of inflammation in wild-type and CD163-/- mice. RESULTS: We describe a subpopulation of bone marrow-resident macrophages (BMRMs) characterized by a high expression of CD163 and functionally distinct from classical bone marrow-derived macrophages. Development of CD163+ BMRMs is strictly dependent on IFN regulatory factor-8. CD163+ BMRMs show a specific transcriptome and cytokine secretion pattern demonstrating a specific immunomodulatory profile of these cells. Accordingly, CD163-/- mice show a stronger inflammation in allergic contact dermatitis, indicating a regulatory role of CD163. However, CD163-/- mice are highly susceptible to S aureus infections, demonstrating the relevance of CD163 for antimicrobial defense as well. CONCLUSIONS: Our data indicate that anti-inflammatory and immunosuppressive mechanisms are not necessarily associated with a decreased antimicrobial activity. In contrast, our data define a novel macrophage population that controls overwhelming inflammation on one hand but is also necessary for an effective control of infections on the other hand.

Glucocorticoids employ the monomeric glucocorticoid receptor to potentiate vitamin D3 and parathyroid hormone-induced osteoclastogenesis.

Glucocorticoid (GC) therapy decreases bone mass and increases the risk of fractures. We investigated interactions between the GC dexamethasone (DEX) and the bone resorptive agents 1,25(OH)2-vitamin D3 (D3) and parathyroid hormone (PTH) on osteoclastogenesis. We observed a synergistic potentiation of osteoclast progenitor cell differentiation and formation of osteoclasts when DEX was added to either D3- or PTH-treated mouse bone marrow cell (BMC) cultures. Cotreatment of DEX with D3 or PTH increased gene encoding calcitonin receptor (Calcr), acid phosphatase 5, tartrate resistant (Acp5), cathepsin K (Ctsk), and TNF superfamily member 11 (Tnfsf11) mRNA, receptor activator of NF-κB ligand protein (RANKL), numbers of osteoclasts on plastic, and pit formation and release of C-terminal fragment of type I collagen from cells cultured on bone slices. Enhanced RANKL protein expression caused by D3 and DEX was absent in BMC from mice in which the GC receptor (GR) was deleted in stromal cells/osteoblasts. Synergistic interactions between DEX and D3 on RANKL and osteoclast formation were present in BMC from mice with attenuated GR dimerization. These data demonstrate that the GR cooperates with D3 and PTH signaling, causing massive osteoclastogenesis, which may explain the rapid bone loss observed with high dosages of GC treatment.-Conaway, H. H., Henning, P., Lie, A., Tuckermann, J., Lerner, U. H. Glucocorticoids employ the monomeric glucocorticoid receptor to potentiate vitamin D3 and parathyroid hormone-induced osteoclastogenesis.

A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

The glucocorticoid receptor (GR) represents the crucial molecular mediator of key endocrine, glucocorticoid hormone-dependent regulatory circuits, including control of glucose, protein, and lipid homeostasis. Consequently, aberrant glucocorticoid signaling is linked to severe metabolic disorders, including insulin resistance, obesity, and hyperglycemia, all of which also appear upon chronic glucocorticoid therapy for the treatment of inflammatory conditions. Of note, long-term glucocorticoid exposure under these therapeutic conditions typically induces glucocorticoid resistance, requiring higher doses and consequently triggering more severe metabolic phenotypes. However, the molecular basis of acquired glucocorticoid resistance remains unknown. In a screen of differential microRNA expression during glucocorticoid-dependent adipogenic differentiation of human multipotent adipose stem cells, we identified microRNA 29a (miR-29a) as one of the most down-regulated transcripts. Overexpression of miR-29a impaired adipogenesis. We found that miR-29a represses GR in human adipogenesis by directly targeting its mRNA, and downstream analyses revealed that GR mediates most of miR-29a's anti-adipogenic effects. Conversely, miR-29a expression depends on GR activation, creating a novel miR-29-driven feedback loop. miR-29a and GR expression were inversely correlated both in murine adipose tissue and in adipose tissue samples obtained from human patients. In the latter, miR-29a levels were additionally strongly negatively correlated with body mass index and adipocyte size. Importantly, inhibition of miR-29 in mice partially rescued the down-regulation of GR during dexamethasone treatment. We discovered that, in addition to modulating GR function under physiologic conditions, pharmacologic glucocorticoid application in inflammatory disease also induced miR-29a expression, correlating with reduced GR levels. This effect was abolished in mice with impaired GR function. In summary, we uncovered a novel GR-miR-29a negative feedback loop conserved between mice and humans, in health and disease. For the first time, we elucidate a microRNA-related mechanism that might contribute to GR dysregulation and resistance in peripheral tissues.-Glantschnig, C., Koenen, M., Gil-Lozano, M., Karbiener, M., Pickrahn, I., Williams-Dautovich, J., Patel, R., Cummins, C. L., Giroud, M., Hartleben, G., Vogl, E., Blüher, M., Tuckermann, J., Uhlenhaut, H., Herzig, S., Scheideler, M. A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

Osteoblast-derived NOTUM reduces cortical bone mass in mice and the NOTUM locus is associated with bone mineral density in humans.

Osteoporosis is a common skeletal disease, affecting millions of individuals worldwide. Currently used osteoporosis treatments substantially reduce vertebral fracture risk, whereas nonvertebral fracture risk, mainly caused by reduced cortical bone mass, has only moderately been improved by the osteoporosis drugs used, defining an unmet medical need. Because several wingless-type MMTV integration site family members (WNTs) and modulators of WNT activity are major regulators of bone mass, we hypothesized that NOTUM, a secreted WNT lipase, might modulate bone mass via an inhibition of WNT activity. To characterize the possible role of endogenous NOTUM as a physiologic modulator of bone mass, we developed global, cell-specific, and inducible Notum-inactivated mouse models. Notum expression was high in the cortical bone in mice, and conditional Notum inactivation revealed that osteoblast lineage cells are the principal source of NOTUM in the cortical bone. Osteoblast lineage-specific Notum inactivation increased cortical bone thickness via an increased periosteal circumference. Inducible Notum inactivation in adult mice increased cortical bone thickness as a result of increased periosteal bone formation, and silencing of Notum expression in cultured osteoblasts enhanced osteoblast differentiation. Large-scale human genetic analyses identified genetic variants mapping to the NOTUM locus that are strongly associated with bone mineral density (BMD) as estimated with quantitative ultrasound in the heel. Thus, osteoblast-derived NOTUM is an essential local physiologic regulator of cortical bone mass via effects on periosteal bone formation in adult mice, and genetic variants in the NOTUM locus are associated with BMD variation in adult humans. Therapies targeting osteoblast-derived NOTUM may prevent nonvertebral fractures.-Movérare-Skrtic, S., Nilsson, K. H., Henning, P., Funck-Brentano, T., Nethander, M., Rivadeneira, F., Coletto Nunes, G., Koskela, A., Tuukkanen, J., Tuckermann, J., Perret, C., Souza, P. P. C., Lerner, U. H., Ohlsson, C. Osteoblast-derived NOTUM reduces cortical bone mass in mice and the NOTUM locus is associated with bone mineral density in humans.

Induced global deletion of glucocorticoid receptor impairs fracture healing.

Although endogenous glucocorticoids (GCs) are important regulators of bone integrity and the immune system, their role in bone repair after fracture-a process highly dependent on inflammation and bone formation-is unclear. Because most effects of GCs are mediated by the glucocorticoid receptor (GR), we used an inducible global GR knockout (GRgtROSACreERT2) mouse model to eliminate endogenous GC action in all cells contributing to bone repair. The healing process was analyzed by cytokine/chemokine multiplex analysis, flow cytometry, histology, gene-expression analysis, microcomputed tomography, and biomechanical analysis. We observed increased early systemic and local inflammatory responses, as well as a significantly higher number of T cells infiltrating the fracture callus. Later in the healing process, we found impaired endochondral ossification in the absence of the GR, leading to persistent cartilage in the calli of the GRgtROSACreERT2 mice, decreased bending stiffness, and a significantly lower proportion of healed bones. Collectively, our data show that the absence of the GR significantly impairs fracture healing associated with a defective cartilage-to-bone transition, underscoring an important role of GCs during fracture healing.-Rapp, A. E., Hachemi, Y., Kemmler, J., Koenen, M., Tuckermann, J., Ignatius, A. Induced global deletion of glucocorticoid receptor impairs fracture healing.