Effects of Systemic Peroxisome Proliferator-Activated Receptor Gamma Inhibition on Bone and Immune Cells in Aged Female Mice.

This study investigates the effects of peroxisome proliferator-activated receptor gamma (PPARγ) inhibition on bone and immune cell profiles in aged female mice, as well as in vitro stromal stem cell osteogenic differentiation and inflammation gene expression. The hypothesis was that inhibition of PPARγ would increase bone mass and alter immune and other cellular functions. Our results showed that treatment with PPARγ antagonist GW9662 for 6 weeks reduced bone volume and trabecular number and increased trabecular spacing. However, inhibition of PPARγ had no significant effect on marrow and spleen immune cell composition in aged female mice. In vitro experiments indicated that GW9662 treatment increased the expression of osteogenic genes but did not affect adipogenic genes. Additionally, GW9662 treatment decreased the expression of several inflammation-related genes. Overall, these findings suggest that PPARγ inhibition may have adverse effects on bone in aged female mice.

Gut microbiota impacts bone via Bacteroides vulgatus-valeric acid-related pathways.

Although the gut microbiota has been reported to influence osteoporosis risk, the individual species involved, and underlying mechanisms, remain largely unknown. We performed integrative analyses in a Chinese cohort of peri-/post-menopausal women with metagenomics/targeted metabolomics/whole-genome sequencing to identify novel microbiome-related biomarkers for bone health. Bacteroides vulgatus was found to be negatively associated with bone mineral density (BMD), which was validated in US white people. Serum valeric acid (VA), a microbiota derived metabolite, was positively associated with BMD and causally downregulated by B. vulgatus. Ovariectomized mice fed B. vulgatus demonstrated increased bone resorption and poorer bone micro-structure, while those fed VA demonstrated reduced bone resorption and better bone micro-structure. VA suppressed RELA protein production (pro-inflammatory), and enhanced IL10 mRNA expression (anti-inflammatory), leading to suppressed maturation of osteoclast-like cells and enhanced maturation of osteoblasts in vitro. The findings suggest that B. vulgatus and VA may represent promising targets for osteoporosis prevention/treatment.

DPP4-Truncated CXCL12 Alters CXCR4/ACKR3 Signaling, Osteogenic Cell Differentiation, Migration, and Senescence.

Bone marrow skeletal stem cells (SSCs) secrete many cytokines including stromal derived factor-1 or CXCL12, which influences cell proliferation, migration, and differentiation. All CXCL12 splice variants are rapidly truncated on their N-terminus by dipeptidyl peptidase 4 (DPP4). This includes the common variant CXCL12 alpha (1-68) releasing a much less studied metabolite CXCL12(3-68). Here, we found that CXCL12(3-68) significantly inhibited SSC osteogenic differentiation and RAW-264.7 cell osteoclastogenic differentiation and induced a senescent phenotype in SSCs. Importantly, pre-incubation of SSCs with CXCL12(3-68) significantly diminished their ability to migrate toward CXCL12(1-68) in transwell migration assays. Using a high-throughput G-protein-coupled receptor (GPCR) screen (GPCRome) and bioluminescent resonance energy transfer molecular interaction assays, we revealed that CXCL12(3-68) acts via the atypical cytokine receptor 3-mediated ß-arrestin recruitment and as a competitive antagonist to CXCR4-mediated signaling. Finally, a reverse phase protein array assay revealed that DPP4-cleaved CXCL12 possesses a different downstream signaling profile from that of intact CXCL12 or controls. The data presented herein provides insights into regulation of CXCL12 signaling. Importantly, it demonstrates that DPP4 proteolysis of CXCL12 generates a metabolite with significantly different and previously overlooked bioactivity that helps explain discrepancies in the literature. This also contributes to an understanding of the molecular mechanisms of osteoporosis and bone fracture repair and could potentially significantly affect the interpretation of experimental outcomes with clinical consequences in other fields where CXCL12 is vital, including cancer biology, immunology, cardiovascular biology, neurobiology, and associated pathologies.

Expression of long noncoding RNA Xist is induced by glucocorticoids.

Glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressive agents. However, their clinical usage is limited by severe multisystemic side effects. Glucocorticoid induced osteoporosis results in significant morbidity and mortality but the cellular and molecular mechanisms underlying GC-induced bone loss are not clear. GC use results in decreased osteoblast differentiation with increased marrow adiposity through effects on bone marrow stem cells. GC effects are transduced through its receptor (GR). To identify novel GR regulated genes, we performed RNA sequencing (RNA-Seq) analysis comparing conditional GR knockout mouse made by crossing the floxed GR animal with the Col I promoter-Cre, versus normal floxed GR without Cre, and that testing was specific for Col I promoter active cells, such as bone marrow mesenchymal stem/osteoprogenitor cells (MSCs) and osteoblasts. Results showed 15 upregulated genes (3- to 10-fold) and 70 downregulated genes (-2.7- to -10-fold), with the long noncoding RNA X-inactive specific transcript (Xist) downregulated the most. The differential expression of genes measured by RNA-Seq was validated by qRT-PCR analysis of selected genes and the GC/GR signaling-dependent expression of Xist was further demonstrated by GC (dexamethasone) treatment of GR-deficient MSCs in vitro and by GC injection of C57BL/6 mice (wild-type males and females) in vivo. Our data revealed that the long noncoding RNA Xist is a GR regulated gene and its expression is induced by GC both in vitro and in vivo. To our knowledge, this is the first evidence showing that Xist is transcriptionally regulated by GC/GR signaling.

The Glucocorticoid Receptor in Osterix-Expressing Cells Regulates Bone Mass, Bone Marrow Adipose Tissue, and Systemic Metabolism in Female Mice During Aging.

Hallmarks of aging-associated osteoporosis include bone loss, bone marrow adipose tissue (BMAT) expansion, and impaired osteoblast function. Endogenous glucocorticoid levels increase with age, and elevated glucocorticoid signaling, associated with chronic stress and dysregulated metabolism, can have a deleterious effect on bone mass. Canonical glucocorticoid signaling through the glucocorticoid receptor (GR) was recently investigated as a mediator of osteoporosis during the stress of chronic caloric restriction. To address the role of the GR in an aging-associated osteoporotic phenotype, the current study utilized female GR conditional knockout (GR-CKO; GRfl/fl :Osx-Cre+) mice and control littermates on the C57BL/6 background aged to 21 months and studied in comparison to young (3- and 6-month-old) mice. GR deficiency in Osx-expressing cells led to low bone mass and BMAT accumulation that persisted with aging. Surprisingly, however, GR-CKO mice also exhibited alterations in muscle mass (reduced % lean mass and soleus fiber size), accompanied by reduced voluntary physical activity, and also exhibited higher whole-body metabolic rate and elevated blood pressure. Moreover, increased lipid storage was observed in GR-CKO osteoblastic cultures in a glucocorticoid-dependent fashion despite genetic deletion of the GR, and could be reversed via pharmacological inhibition of the mineralocorticoid receptor (MR). These findings provide evidence of a role for the GR (and possibly the MR) in facilitating healthy bone maintenance with aging in females. The effects of GR-deficient bone on whole-body physiology also demonstrate the importance of bone as an endocrine organ and suggest evidence for compensatory mechanisms that facilitate glucocorticoid signaling in the absence of osteoblastic GR function; these represent new avenues of research that may improve understanding of glucocorticoid signaling in bone toward the development of novel osteogenic agents. © 2021 American Society for Bone and Mineral Research (ASBMR).

Age-associated changes in microRNAs affect the differentiation potential of human mesenchymal stem cells: Novel role of miR-29b-1-5p expression.

Age-associated osteoporosis is widely accepted as involving the disruption of osteogenic stem cell populations and their functioning. Maintenance of the local bone marrow (BM) microenvironment is critical for regulating proliferation and differentiation of the multipotent BM mesenchymal stromal/stem cell (BMSC) population with age. The potential role of microRNAs (miRNAs) in modulating BMSCs and the BM microenvironment has recently gained attention. However, miRNAs expressed in rapidly isolated BMSCs that are naïve to the non-physiologic standard tissue culture conditions and reflect a more accurate in vivo profile have not yet been reported. Here we directly isolated CD271 positive (+) BMSCs within hours from human surgical BM aspirates without culturing and performed microarray analysis to identify the age-associated changes in BMSC miRNA expression. One hundred and two miRNAs showed differential expression with aging. Target prediction and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the up-regulated miRNAs targeting genes in bone development pathways were considerably enriched. Among the differentially up-regulated miRNAs the novel passenger strand miR-29b-1-5p was abundantly expressed as a mature functional miRNA with aging. This suggests a critical arm-switching mechanism regulates the expression of the miR-29b-1-5p/3p pair shifting the normally degraded arm, miR-29b-1-5p, to be the dominantly expressed miRNA of the pair in aging. The normal guide strand miR-29b-1-3p is known to act as a pro-osteogenic miRNA. On the other hand, overexpression of the passenger strand miR-29b-1-5p in culture-expanded CD271+ BMSCs significantly down-regulated the expression of stromal cell-derived factor 1 (CXCL12)/ C-X-C chemokine receptor type 4 (SDF-1(CXCL12)/CXCR4) axis and other osteogenic genes including bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (RUNX2). In contrast, blocking of miR-29b-1-5p function using an antagomir inhibitor up-regulated expression of BMP-2 and RUNX2 genes. Functional assays confirmed that miR-29b-1-5p negatively regulates BMSC osteogenesis in vitro. These novel findings provide evidence of a pathogenic anti-osteogenic role for miR-29b-1-5p and other miRNAs in age-related defects in osteogenesis and bone regeneration.

Photobiomodulation has rejuvenating effects on aged bone marrow mesenchymal stem cells.

The plasticity and proliferative capacity of stem cells decrease with aging, compromising their tissue regenerative potential and therapeutic applications. This decline is directly linked to mitochondrial dysfunction. Here, we present an effective strategy to reverse aging of mouse bone marrow mesenchymal stem cells (BM-MSCs) by restoring their mitochondrial functionality using photobiomodulation (PBM) therapy. Following the characterization of young and aged MSCs, our results show that a near-infrared PBM treatment delivering 3 J/cm2 is the most effective modality for improving mitochondrial functionality and aging markers. Furthermore, our results unveil that young and aged MSCs respond differently to the same modality of PBM: whereas the beneficial effect of a single PBM treatment dissipates within 7 h in aged stem cells, it is lasting in young ones. Nevertheless, by applying three consecutive treatments at 24-h intervals, we were able to obtain a lasting rejuvenating effect on aged MSCs. Our findings are of particular significance for improving autologous stem cell transplantation in older individuals who need such therapies most.

Ameliorative Effects of Component Chinese Medicine From Curcumae Rhizoma and Sparganii Rhizoma, a Traditional Herb Pair, on Uterine Leiomyoma in a Rat Model.

Uterine leiomyoma (UL), common benign tumors in women of child-bearing age, are believed to be caused mainly by Qi stagnation and blood stasis, according to a theory of traditional Chinese medicine. Curcumae Rhizoma and Sparganii Rhizoma (CRSR) is a classical herb pair that activates blood circulation to dissipate blood stasis. The purpose of this study was to explore the prevention and treatment effects of CRSR component compatibility on UL in rats. We randomly assigned adult female non-pregnant rats into three groups: a normal control (NC) group, a UL model group, and a CRSR treatment group. We administered to the UL and CRSR groups oral gavage diethylstilbestrol and injected them with progesterone (P) to establish UL for 5 weeks. The CRSR group received a CRSR medicinal solution after daily modeling. The uterus morphology of the UL group showed significantly more swelling than did that of the NC group, and we found no significant abnormalities in the morphology of the CRSR group. The pathological changes associated with UL were relieved in the CRSR group. CRSR improved the related parameters of the uterus and ovarian coefficients, significantly reducing the concentrations of P in the serum and the concentrations of estradiol, P, estrogen receptor, and P receptor in the uterus and ovary. In addition, CRSR significantly improved the abnormal blood conditions of UL, shown by decreases in plasma viscosity, the erythrocyte sedimentation rate equation K value, and erythrocyte aggregation index. Therefore, CRSR component compatibility may prevent and cure UL through the above ways.

Deficiency of PPARγ in Bone Marrow Stromal Cells Does not Prevent High-Fat Diet-Induced Bone Deterioration in Mice.

BACKGROUND: Bone marrow osteoblasts and adipocytes are derived from a common mesenchymal stem cell and have a reciprocal relationship. Peroxisome proliferator-activated receptor gamma (PPARγ), a regulator for adipocyte differentiation, may be a potential target for reducing obesity and increasing bone mass. OBJECTIVES: This study tested the hypothesis that bone-specific Pparg conditional knockout (cKO), via deletion of Pparg from bone marrow stromal cells (BMSC) using Osterix 1 (Osx1)-Cre, would prevent high-fat (HF) diet-induced bone deterioration in mice. METHODS: PPARγ cKO (PPARγfl/fl: Osx1-Cre) and floxed littermate control (PPARγfl/fl Osx1-Cre- ) mice that were 6 weeks old were randomly assigned to 4 groups (n = 12/group, 6 male and 6 female) and fed ad libitum with either a normal-fat (NF) purified diet (3.85 kcal/g; 10% energy as fat) or an HF diet (4.73 kcal/g; 45% energy as fat) for 6 mo. Bone structure, body composition, and serum bone-related cytokines were measured. Data were analyzed by 2-way ANOVA with Tukey post hoc comparison. RESULTS: The HF diet decreased the tibial and lumbar vertebrae trabecular bone volume/total volume (BV/TV) by 28% and 18%, respectively, compared to the NF diet (P < 0.01). PPARγ cKO mice had 23% lower body fat mass and 9% lower lean mass than control mice. PPARγ cKO mice had 41% greater tibial trabecular BV/TV compared to control mice. None of trabecular bone parameters at the second lumbar vertebra were affected by genotype. PPARγ cKO mice had decreased cortical thickness compared to control mice. PPARγ cKO mice had a 14% lower (P < 0.01) serum concentration of leptin and a 35% higher (P < 0.05) concentration of osteocalcin compared with control mice. CONCLUSIONS: These data indicate that PPARγ has site-specific impacts on bone structures in mice and that knockout PPARγ in BMSC increased bone mass (BV/TV) in the tibia but not the lumbar vertebrae. PPARγ disruption in BMSC did not prevent HF diet-induced bone deterioration in mice.

Inhibition of Fibroblast Activation in Uterine Leiomyoma by Components of Rhizoma Curcumae and Rhizoma Sparganii.

Background: The herbs Rhizoma Curcumae and Rhizoma Sparganii (RCRS) are often used in traditional Chinese medicine for the treatment of uterine leiomyoma (UL). The effectiveness of RCRS for the treatment of UL has been confirmed in our previous studies. Purpose: This study aimed to investigate the molecular mechanism by which RCRS inhibits the activation of fibroblast activation protein (FAP) and prevents UL in rats. Study Design and Methods: A Sprague Dawley (SD) rat model of UL was established via estrogen and progesterone load combined with external stimulation. Histological analyses, enzyme-linked immunosorbent assays, and western blotting were performed to evaluate the effect of RCRS on UL and elucidate its mechanism of action. Results: Our data showed that the treatment of SD rats with RCRS significantly reduced the expression of extracellular matrix component collagen, FAP, and transforming growth factor beta (a FAP-activating factor) and the phosphorylation of the cell proliferation pathway-related signaling factors AKT/MEK/ERK. Conclusion: Our results suggest that RCRS is effective in the prevention and treatment of UL in rats, and RCRS may exert its functions by inhibiting the activation of tumor-associated fibroblasts and cell proliferation and by improving the tumor extracellular matrix.

Kynurenine Promotes RANKL-Induced Osteoclastogenesis In Vitro by Activating the Aryl Hydrocarbon Receptor Pathway.

There is increasing evidence of the involvement of the tryptophan metabolite kynurenine (KYN) in disrupting osteogenesis and contributing to aging-related bone loss. Here, we show that KYN has an effect on bone resorption by increasing osteoclastogenesis. We have previously reported that in vivo treatment with KYN significantly increased osteoclast number lining bone surfaces. Here, we report the direct effect of KYN on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in Raw 264.7 macrophage cells, and we propose a potential mechanism for these KYN-mediated effects. We show that KYN/RANKL treatment results in enhancement of RANKL-induced osteoclast differentiation. KYN drives upregulation and activation of the key osteoclast transcription factors, c-fos and NFATc1 resulting in an increase in the number of multinucleated TRAP+ osteoclasts, and in hydroxyapatite bone resorptive activity. Mechanistically, the KYN receptor, aryl hydrocarbon receptor (AhR), plays an important role in the induction of osteoclastogenesis. We show that blocking AhR signaling using an AhR antagonist, or AhR siRNA, downregulates the KYN/RANKL-mediated increase in c-fos and NFATc1 and inhibits the formation of multinucleated TRAP + osteoclasts. Altogether, this work highlights that the novelty of the KYN and AhR pathways might have a potential role in helping to regulate osteoclast function with age and supports pursuing additional research to determine if they are potential therapeutic targets for the prevention or treatment of osteoporosis.

Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells.

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis.

Picolinic acid, a tryptophan oxidation product, does not impact bone mineral density but increases marrow adiposity.

Tryptophan is an essential amino acid catabolized initially to kynurenine (kyn), an immunomodulatory metabolite that we have previously shown to promote bone loss. Kyn levels increase with aging and have also been associated with neurodegenerative disorders. Picolinic acid (PA) is another tryptophan metabolite downstream of kyn. However, in contrast to kyn, PA is reported to be neuroprotective and further, to promote osteogenesis in vitro. Thus, we hypothesized that PA might be osteoprotective in vivo. In an IACUC-approved protocol, we fed PA to aged (23-month-old) C57BL/6 mice for eight weeks. In an effort to determine potential interactions of PA with dietary protein we also fed PA in a low-protein diet (8%). The mice were divided into four groups: Control (18% dietary protein), +PA (700 ppm); Low-protein (8%), +PA (700 ppm). The PA feedings had no impact on mouse weight, body composition or bone density. At sacrifice bone and stem cells were collected for analysis, including µCT and RT-qPCR. Addition of PA to the diet had no impact on trabecular bone parameters. However, marrow adiposity was significantly increased in PA-fed mice, and in bone marrow stromal cells isolated from these mice increases in the expression of the lipid storage genes, Plin1 and Cidec, were observed. Thus, as a downstream metabolite of kyn, PA no longer showed kyn's detrimental effects on bone but instead appears to impact energy balance.

Deletion of PPARγ in Mesenchymal Lineage Cells Protects Against Aging-Induced Cortical Bone Loss in Mice.

Bone loss in aging is linked with chronic low-grade inflammation and the accumulation of marrowfat in animals and humans. Peroxisome proliferator-activated receptor gamma (PPARγ), an adipogenic regulator, plays key roles in these biological processes. However, studies of the roles of PPARγ in age-related bone loss and inflammation are lacking. We hypothesized that deletion of PPARγ in bone marrow mesenchymal lineage cells would reduce bone loss with aging, potentially through a reduction in fat-generated inflammatory responses and an increase in osteoblastic activity. In the present study, we show that mice deficient of PPARγ in Dermo1-expressing mesenchymal lineage cells (Dermo1-Cre:PPARγ fl/fl) have reduced fat mass and increased cortical bone thickness but that deficiency of PPARγ had limited effect on protection of trabecular bone with aging as demonstrated by dual-energy X-ray absorptiometry, µCT, and histomorphometric analyses. Conditional knockout of PPARγ reduced serum concentrations of adipokines, including adiponectin, resistin, and leptin, and reduced marrow stromal cell expression levels of inflammation-related genes. Inflammation genes involved in the interferon signaling pathway were reduced the most. These results demonstrate that disruption of the master adipogenic regulator, PPARγ, has a certain protective effect on aging-induced bone loss, suggesting that regulation of adipose function and modulation of interferon signaling are among the key mechanisms by which PPARγ regulates bone homeostasis during aging process.

Kynurenine inhibits autophagy and promotes senescence in aged bone marrow mesenchymal stem cells through the aryl hydrocarbon receptor pathway.

Osteoporosis is an age-related deterioration in bone health that is, at least in part, a stem cell disease. The different mechanisms and signaling pathways that change with age and contribute to the development of osteoporosis are being identified. One key upstream mechanism that appears to target a number of osteogenic pathways with age is kynurenine, a tryptophan metabolite and an endogenous Aryl hydrocarbon receptor (AhR) agonist. The AhR signaling pathway has been reported to promote aging phenotypes across species and in different tissues. We previously found that kynurenine accumulates with age in the plasma and various tissues including bone and induces bone loss and osteoporosis in mice. Bone marrow mesenchymal stem cells (BMSCs) are responsible for osteogenesis, adipogenesis, and overall bone regeneration. In the present study, we investigated the effect of kynurenine on BMSCs, with a focus on autophagy and senescence as two cellular processes that control BMSCs proliferation and differentiation capacity. We found that physiological levels of kynurenine (10 and 100 µM) disrupted autophagic flux as evidenced by the reduction of LC3B-II, and autophagolysosomal production, as well as a significant increase of p62 protein level. Additionally, kynurenine also induced a senescent phenotype in BMSCs as shown by the increased expression of several senescence markers including senescence associated ß-galactosidase in BMSCs. Additionally, western blotting reveals that levels of p21, another marker of senescence, also increased in kynurenine-treated BMSCs, while senescent-associated aggregation of nuclear H3K9me3 also showed a significant increase in response to kynurenine treatment. To validate that these effects are in fact due to AhR signaling pathway, we utilized two known AhR antagonists: CH-223191, and 3',4'-dimethoxyflavone to try to block AhR signaling and rescue kynurenine /AhR mediated effects. Indeed, AhR inhibition restored kynurenine-suppressed autophagy levels as shown by levels of LC3B-II, p62 and autophagolysosomal formation demonstrating a rescuing of autophagic flux. Furthermore, inhibition of AhR signaling prevented the kynurenine-induced increase in senescence associated ß-galactosidase and p21 levels, as well as blocking aggregation of nuclear H3K9me3. Taken together, our results suggest that kynurenine inhibits autophagy and induces senescence in BMSCs via AhR signaling, and that this may be a novel target to prevent or reduce age-associated bone loss and osteoporosis.

Endogenous Glucocorticoid Signaling in the Regulation of Bone and Marrow Adiposity: Lessons from Metabolism and Cross Talk in Other Tissues.

PURPOSE OF REVIEW: The development of adiposity in the bone marrow, known as marrow adipose tissue (MAT), is often associated with musculoskeletal frailty. Glucocorticoids, which are a key component of the biological response to stress, affect both bone and MAT. These molecules signal through receptors such as the glucocorticoid receptor (GR), but the role of the GR in regulation of MAT is not yet clear from previous studies. The purpose of this review is to establish and determine the role of GR-mediated signaling in marrow adiposity by comparing and contrasting what is known against other energy-storing tissues like adipose tissue, liver, and muscle, to provide better insight into the regulation of MAT during times of metabolic stress (e.g., dietary challenges, aging). RECENT FINDINGS: GR-mediated glucocorticoid signaling is critical for proper storage and utilization of lipids in cells such as adipocytes and hepatocytes and proteolysis in muscle, impacting whole-body composition, energy utilization, and homeostasis through a complex network of tissue cross talk between these systems. Loss of GR signaling in bone promotes increased MAT and decreased bone mass. GR-mediated signaling in the liver, adipose tissue, and muscle is critical for whole-body energy and metabolic homeostasis, and both similarities and differences in GR-mediated GC signaling in MAT as compared with these tissues are readily apparent. It is clear that GC-induced pathways work together through these tissues to affect systemic biology, and understanding the role of bone in these patterns of tissue cross talk may lead to a better understanding of MAT-bone biology that improves treatment strategies for frailty-associated diseases.

Amino acids as signaling molecules modulating bone turnover.

Except for the essential amino acids (AAs), much of the focus on adequate dietary protein intake has been on total nitrogen and caloric intake rather than AA composition. Recent data, however, demonstrate that "amino-acid sensing" can occur through either intracellular or extracellular nutrient-sensing mechanisms. In particular, members of the class 3 G-protein coupled receptor family, like the calcium-sensing receptor are known to preferentially bind specific AAs, which then modulate receptor activation by calcium ions and thus potentially impact bone turnover. In pursuing the possibility of direct nutrient effects on bone cells, we examined individual AA effects on osteoprogenitor/bone marrow stromal cells (BMSCs), a key target for bone anabolism. We demonstrate that BMSCs express both intracellular and extracellular nutrient sensing pathways and that AAs are required for BMSC survival. In addition, certain AA types, like members of the aromatic AAs, can potently stimulate increases in intracellular calcium and ERK phosphorylation/activation. Further, based on the in vitro data, we examined the effect of specific AAs on bone mass. To better evaluate the impact of specific AAs, we added these to a low-protein diet. Our data demonstrate that a low-protein diet itself is associated with a significant drop in bone mineral density (BMD) in the older mice, related, at least in part, to an increase in osteoclastic activity. This drop in BMD in mice on the low-protein diet is prevented by addition of AAs from the aromatic group. Taken together our data show that AAs function as specific and selective signaling molecules in bone cells.

Deletion of protein kinase D1 in osteoprogenitor cells results in decreased osteogenesis in vitro and reduced bone mineral density in vivo.

Protein kinase D1 (PRKD1) is thought to play a role in a number of cellular functions, including proliferation and differentiation. We hypothesized that PRKD1 in bone marrow-derived mesenchymal stem cells (BMMSC) could modulate osteogenesis. In BMMSCs from floxed PRKD1 mice, PRKD1 ablation with adenovirus-mediated Cre-recombinase expression inhibited BMMSC differentiation in vitro. In 3- and 6-month-old conditional knockout mice (cKO), in which PRKD1 was ablated in osteoprogenitor cells by osterix promoter-driven Cre-recombinase, bone mineral density (BMD) was significantly reduced compared with floxed control littermates. Microcomputed tomography analysis also demonstrated a decrease in trabecular thickness and bone volume fraction in cKO mice at these ages. Dynamic bone histomorphometry suggested a mineralization defect in the cKO mice. However, by 9 months of age, the bone appeared to compensate for the lack of PRKD1, and BMD was not different. Taken together, these results suggest a potentially important role for PRKD1 in bone formation.

Differentially expressed genes in PPARγ-deficient MSCs.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipogenesis. It is also a central player in energy metabolism, inflammation and immunity. As an important nuclear transcription factor, PPARγ can regulate the expression and function of genes or biological processes directly or indirectly via association with other factors and thus modulate their activities. To better understand the impact of PPARγ on the global gene expression profile, we evaluated the bioinformatic data, which revealed the changes that occurred in genes and their pathways in the absence of PPARγ. In brief, we performed RNA deep sequencing (RNA-Seq) analysis using RNA samples isolated from multipotent mesenchymal stromal cells (MSCs) of PPARγ knockout and wild type control mice. The RNA-Seq data sets were then subjected to bioinformatic analyses from various angles to better reveal the breadth of PPARγ function in different biological processes. Our results reveal novel genes and networks modulated by PPARγ and provides new insights into our understanding of the physiologic and pathophysiologic role this nuclear receptor plays in health and disease.

Role of glucocorticoid-induced leucine zipper (GILZ) in inflammatory bone loss.

TNF-α plays a key role in the development of rheumatoid arthritis (RA) and inflammatory bone loss. Unfortunately, treatment of RA with anti-inflammatory glucocorticoids (GCs) also causes bone loss resulting in osteoporosis. Our previous studies showed that overexpression of glucocorticoid-induced leucine zipper (GILZ), a mediator of GC's anti-inflammatory effect, can enhance osteogenic differentiation in vitro and bone acquisition in vivo. To investigate whether GILZ could antagonize TNF-α-induced arthritic inflammation and protect bone in mice, we generated a TNF-α-GILZ double transgenic mouse line (TNF-GILZ Tg) by crossbreeding a TNF-α Tg mouse, which ubiquitously expresses human TNF-α, with a GILZ Tg mouse, which expresses mouse GILZ under the control of a 3.6kb rat type I collagen promoter fragment. Results showed that overexpression of GILZ in bone marrow mesenchymal stem/progenitor cells protected mice from TNF-α-induced inflammatory bone loss and improved bone integrity (TNF-GILZ double Tg vs. TNF-αTg, n = 12-15). However, mesenchymal cell lineage restricted GILZ expression had limited effects on TNF-α-induced arthritic inflammation as indicated by clinical scores and serum levels of inflammatory cytokines and chemokines.