The Role of Aryl Hydrocarbon Receptor in Bone Biology.

Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is crucial in maintaining the skeletal system. Our study focuses on encapsulating the role of AhR in bone biology and identifying novel signaling pathways in musculoskeletal pathologies using the GEO dataset. The GEO2R analysis identified 8 genes (CYP1C1, SULT6B1, CYB5A, EDN1, CXCR4B, CTGFA, TIPARP, and CXXC5A) involved in the AhR pathway, which play a pivotal role in bone remodeling. The AhR knockout in hematopoietic stem cells showed alteration in several novel bone-related transcriptomes (eg, Defb14, ZNF 51, and Chrm5). Gene Ontology Enrichment Analysis demonstrated 54 different biological processes associated with bone homeostasis. Mainly, these processes include bone morphogenesis, bone development, bone trabeculae formation, bone resorption, bone maturation, bone mineralization, and bone marrow development. Employing Functional Annotation and Clustering through DAVID, we further uncovered the involvement of the xenobiotic metabolic process, p450 pathway, oxidation-reduction, and nitric oxide biosynthesis process in the AhR signaling pathway. The conflicting evidence of current research of AhR signaling on bone (positive and negative effects) homeostasis may be due to variations in ligand binding affinity, binding sites, half-life, chemical structure, and other unknown factors. In summary, our study provides a comprehensive understanding of the underlying mechanisms of the AhR pathway in bone biology.

Circulating Osteoprogenitor Cells Have a Mixed Immune and Mesenchymal Progenitor Function in Humans.

BACKGROUND: Circulating osteoprogenitors (COP) are a population of cells in the peripheral circulation that possess functional and phenotypical characteristics of multipotent stromal cells (MSCs). This population has a solid potential to become an abundant, accessible, and replenishable source of MSCs with multiple potential clinical applications. However, a comprehensive functional characterization of COP cells is still required to test and fully develop their use in clinical settings. METHODS: This study characterized COP cells by comparing them to bone marrow-derived MSCs (BM-MSCs) and adipose-derived MSCs (ASCs) through detailed transcriptomic and proteomic analyses. RESULTS: We demonstrate that COP cells have a distinct gene and protein expression pattern with a significantly stronger immune footprint, likely owing to their hematopoietic lineage. In addition, regarding progenitor cell differentiation and proliferation pathways, COP cells have a similar expression pattern to BM-MSCs and ASCs. CONCLUSION: COP cells are a unique but functionally similar population to BM-MSCs and ASCs, sharing their proliferation and differentiation capacity, thus presenting an accessible source of MSCs with strong potential for translational regenerative medicine strategies.

Inhibiting MicroRNA-141-3p Improves Musculoskeletal Health in Aged Mice.

Emerging evidence shows that the microRNA-141-3p is involved in various age-related pathologies. Previously, our group and others reported elevated levels of miR-141-3p in several tissues and organs with age. Here, we inhibited the expression of miR-141-3p using antagomir (Anti-miR-141-3p) in aged mice and explored its role in healthy aging. We analyzed serum (cytokine profiling), spleen (immune profiling), and overall musculoskeletal phenotype. We found decreased levels of pro-inflammatory cytokines (such as TNF-α, IL-1ß, IFN-γ) in serum with Anti-miR-141-3p treatment. The flow-cytometry analysis on splenocytes revealed decreased M1 (pro-inflammatory) and increased M2 (anti-inflammatory) populations. We also found improved bone microstructure and muscle fiber size with Anti-miR-141-3p treatment. Molecular analysis revealed that miR-141-3p regulates the expression of AU-rich RNA-binding factor 1 (AUF1) and promotes senescence (p21, p16) and pro-inflammatory (TNF-α, IL-1ß, IFN-γ) environment whereas inhibiting miR-141-3p prevents these effects. Furthermore, we demonstrated that the expression of FOXO-1 transcription factor was reduced with Anti-miR-141-3p and elevated with silencing of AUF1 (siRNA-AUF1), suggesting crosstalk between miR-141-3p and FOXO-1. Overall, our proof-of-concept study demonstrates that inhibiting miR-141-3p could be a potential strategy to improve immune, bone, and muscle health with age.

Orchiectomy sensitizes cortical bone in male mice to the harmful effects of kynurenine.

Kynurenine (Kyn) is a tryptophan metabolite that increases with age and promotes musculoskeletal dysfunction. We previously found a sexually dimorphic pattern in how Kyn affects bone, with harmful effects more prevalent in females than males. This raises the possibility that male sex steroids might exert a protective effect that blunts the effects of Kyn in males. To test this, orchiectomy (ORX) or sham surgeries were performed on 6-month-old C57BL/6 mice, after which mice received Kyn (10 mg/kg) or vehicle via intraperitoneal injection, once daily, 5×/week, for four weeks. Bone histomorphometry, DXA, microCT, and serum marker analyses were performed after sacrifice. In vitro studies were performed to specifically test the effect of testosterone on activation of aryl hydrocarbon receptor (AhR)-mediated signaling by Kyn in mesenchymal-lineage cells. Kyn treatment reduced cortical bone mass in ORX- but not sham-operated mice. Trabecular bone was unaffected. Kyn's effects on cortical bone in ORX mice were attributed primarily to enhanced endosteal bone resorption activity. Bone marrow adipose tissue was increased in Kyn-treated ORX animals but was unchanged by Kyn in sham-operated mice. ORX surgery increased mRNA expression of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1a1 in the bone, suggesting a priming and/or amplification of AhR signaling pathways. Mechanistic in vitro studies revealed that testosterone blunted Kyn-stimulated AhR transcriptional activity and Cyp1a1 expression in mesenchymal-linage cells. These data suggest a protective role for male sex steroids in blunting the harmful effects of Kyn in cortical bone. Therefore, testosterone may play an important role in regulating Kyn/AhR signaling in musculoskeletal tissues, suggesting crosstalk between male sex steroids and Kyn signaling may influence age-associated musculoskeletal frailty.

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.

Upregulation of the EGFR/MEK1/MAPK1/2 signaling axis as a mechanism of resistance to antiestrogen­induced BimEL dependent apoptosis in ER+ breast cancer cells.

The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimEL­dependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the pro­apoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL pro­apoptotic action in an antiestrogen­resistant breast cancer cell model. In addition, the present study identified a pro­survival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogen­resistant breast cancer cells survive EGFR targeting by pro­survival autophagy. These pre­clinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and pro­survival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimEL­dependent apoptosis.

Minocycline-induced disruption of the intestinal FXR/FGF15 axis impairs osteogenesis in mice.

Antibiotic-induced shifts in the indigenous gut microbiota influence normal skeletal maturation. Current theory implies that gut microbiota actions on bone occur through a direct gut/bone signaling axis. However, our prior work supports that a gut/liver signaling axis contributes to gut microbiota effects on bone. Our purpose was to investigate the effects of minocycline, a systemic antibiotic treatment for adolescent acne, on pubertal/postpubertal skeletal maturation. Sex-matched specific pathogen-free (SPF) and germ-free (GF) C57BL/6T mice were administered a clinically relevant minocycline dose from age 6-12 weeks. Minocycline caused dysbiotic shifts in the gut bacteriome and impaired skeletal maturation in SPF mice but did not alter the skeletal phenotype in GF mice. Minocycline administration in SPF mice disrupted the intestinal farnesoid X receptor/fibroblast growth factor 15 axis, a gut/liver endocrine axis supporting systemic bile acid homeostasis. Minocycline-treated SPF mice had increased serum conjugated bile acids that were farnesoid X receptor (FXR) antagonists, suppressed osteoblast function, decreased bone mass, and impaired bone microarchitecture and fracture resistance. Stimulating osteoblasts with the serum bile acid profile from minocycline-treated SPF mice recapitulated the suppressed osteogenic phenotype found in vivo, which was mediated through attenuated FXR signaling. This work introduces bile acids as a potentially novel mediator of gut/liver signaling actions contributing to gut microbiota effects on bone.

Alterations in bone metabolites with age in C57BL/6 mice model.

Understanding the pathophysiology behind age-related diseases is an urgent need as the elderly population continues to grow. With age, there is a high risk of musculoskeletal deterioration and associated morbidity and mortality. Although the exact mechanism behind age-related degeneration is unknown, it is well established that alteration in cellular metabolism is one of the important contributing factors. Alteration in signaling pathways with age leads to the accumulation or depletion of several metabolites that play a vital role in musculoskeletal pathophysiology. This study aimed to identify age-related changes in bone tissue metabolites in C57BL/6 mice. We then correlated the differentially expressed metabolites with their functions in bone biology. In both aged males and females, hydroxyproline, glutamine, and alpha-linolenic acid levels were decreased. In aged females, Ornithine (p value = 0.001), L-Proline (p value = 0.008), Uridine (p value = 0.001), Aspartic Acid (p value = 0.004) levels were significantly decreased, and glutamate (p value = 0.002) was elevated. In aged males, N-acetyl-D-glucosamine (pvalue = 0.010), Adrenic acid (pvalue = 0.0099), Arachidonic acid (p value = 0.029) and Allantoin (p value = 0.004) levels were decreased. Metabolic pathway analysis revealed that purine and D-glutamine and D-glutamate metabolism were significantly altered in both sexes, while arginine biosynthesis in females and lipid metabolism in males were highly affected. These differences in metabolic signaling might be one of the reasons for the discrepancy in musculoskeletal disease manifestation between the two sexes. Understanding the role of these metabolites play in the aging bone will allow for new sex-specific targeted therapies against the progression of musculoskeletal diseases.

Aryl hydrocarbon receptor (AhR)-mediated signaling as a critical regulator of skeletal cell biology.

The aryl hydrocarbon receptor (AhR) has been implicated in regulating skeletal progenitor cells and the activity of bone-forming osteoblasts and bone-resorbing osteoclasts, thereby impacting bone mass and the risk of skeletal fractures. The AhR also plays an important role in the immune system within the skeletal niche and in the differentiation of mesenchymal stem cells into other cell lineages including chondrocytes and adipocytes. This transcription factor responds to environmental pollutants which can act as AhR ligands, initiating or interfering with various signaling cascades to mediate downstream effects, and also responds to endogenous ligands including tryptophan metabolites. This review comprehensively describes the reported roles of the AhR in skeletal cell biology, focusing on mesenchymal stem cells, osteoblasts, and osteoclasts, and discusses how AhR exhibits sexually dimorphic effects in bone. The molecular mechanisms mediating AhR's downstream effects are highlighted to emphasize the potential importance of targeting this signaling cascade in skeletal disorders.

Tryptophan-Kynurenine Pathway in COVID-19-Dependent Musculoskeletal Pathology: A Minireview.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), affecting multiple organ systems, including the respiratory tract and lungs. Several studies have reported that the tryptophan-kynurenine pathway is altered in COVID-19 patients. The tryptophan-kynurenine pathway plays a vital role in regulating inflammation, metabolism, immune responses, and musculoskeletal system biology. In this minireview, we surmise the effects of the kynurenine pathway in COVID-19 patients and how this pathway might impact muscle and bone biology.

Characterization of Differentially Expressed miRNAs by CXCL12/SDF-1 in Human Bone Marrow Stromal Cells.

Stromal cell-derived factor 1 (SDF-1) is known to influence bone marrow stromal cell (BMSC) migration, osteogenic differentiation, and fracture healing. We hypothesize that SDF-1 mediates some of its effects on BMSCs through epigenetic regulation, specifically via microRNAs (miRNAs). MiRNAs are small non-coding RNAs that target specific mRNA and prevent their translation. We performed global miRNA analysis and determined several miRNAs were differentially expressed in response to SDF-1 treatment. Gene Expression Omnibus (GEO) dataset analysis showed that these miRNAs play an important role in osteogenic differentiation and fracture healing. KEGG and GO analysis indicated that SDF-1 dependent miRNAs changes affect multiple cellular pathways, including fatty acid biosynthesis, thyroid hormone signaling, and mucin-type O-glycan biosynthesis pathways. Furthermore, bioinformatics analysis showed several miRNAs target genes related to stem cell migration and differentiation. This study's findings indicated that SDF-1 induces some of its effects on BMSCs function through miRNA regulation.

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.

A Tryptophan-Deficient Diet Induces Gut Microbiota Dysbiosis and Increases Systemic Inflammation in Aged Mice.

The gut microflora is a vital component of the gastrointestinal (GI) system that regulates local and systemic immunity, inflammatory response, the digestive system, and overall health. Older people commonly suffer from inadequate nutrition or poor diets, which could potentially alter the gut microbiota. The essential amino acid (AA) tryptophan (TRP) is a vital diet component that plays a critical role in physiological stress responses, neuropsychiatric health, oxidative systems, inflammatory responses, and GI health. The present study investigates the relationship between varied TRP diets, the gut microbiome, and inflammatory responses in an aged mouse model. We fed aged mice either a TRP-deficient (0.1%), TRP-recommended (0.2%), or high-TRP (1.25%) diet for eight weeks and observed changes in the gut bacterial environment and the inflammatory responses via cytokine analysis (IL-1a, IL-6, IL-17A, and IL-27). The mice on the TRP-deficient diets showed changes in their bacterial abundance of Coriobacteriia class, Acetatifactor genus, Lachnospiraceae family, Enterococcus faecalis species, Clostridium sp genus, and Oscillibacter genus. Further, these mice showed significant increases in IL-6, IL-17A, and IL-1a and decreased IL-27 levels. These data suggest a direct association between dietary TRP content, the gut microbiota microenvironment, and inflammatory responses in aged mice models.

Kynurenine induces an age-related phenotype in bone marrow stromal cells.

Advanced age is one of the important contributing factors for musculoskeletal deterioration. Although the exact mechanism behind this degeneration is unknown, it has been previously established that nutritional signaling plays a vital role in musculoskeletal pathophysiology. Our group established the vital role of the essential amino acid, tryptophan, in aging musculoskeletal health. With advanced age, inflammatory factors activate indoleamine 2,3-dioxygenase (IDO1) and accumulate excessive intermediate tryptophan metabolites such as Kynurenine (KYN). With age, Kynurenine accumulates and suppresses osteogenic differentiation, impairs autophagy, promotes early senescence, and alters cellular bioenergetics of bone marrow stem cells. Recent studies have shown that Kynurenine negatively impacts bone marrow stromal cells (BMSCs) and, consequently, promotes bone loss. Overall, understanding the mechanism behind BMSCs losing their ability for osteogenic differentiation can provide insight into the prevention of osteoporosis and the development of targeted therapies. Therefore, in this article, we review Kynurenine and how it plays a vital role in BMSC dysfunction and bone loss with age.

Modulation of p75NTR on Mesenchymal Stem Cells Increases Their Vascular Protection in Retinal Ischemia-Reperfusion Mouse Model.

Mesenchymal stem cells (MSCs) are a promising therapy to improve vascular repair, yet their role in ischemic retinopathy is not fully understood. The aim of this study is to investigate the impact of modulating the neurotrophin receptor; p75NTR on the vascular protection of MSCs in an acute model of retinal ischemia/reperfusion (I/R). Wild type (WT) and p75NTR-/- mice were subjected to I/R injury by increasing intra-ocular pressure to 120 mmHg for 45 min, followed by perfusion. Murine GFP-labeled MSCs (100,000 cells/eye) were injected intravitreally 2 days post-I/R and vascular homing was assessed 1 week later. Acellular capillaries were counted using trypsin digest 10-days post-I/R. In vitro, MSC-p75NTR was modulated either genetically using siRNA or pharmacologically using the p75NTR modulator; LM11A-31, and conditioned media were co-cultured with human retinal endothelial cells (HREs) to examine the angiogenic response. Finally, visual function in mice undergoing retinal I/R and receiving LM11A-31 was assessed by visual-clue water-maze test. I/R significantly increased the number of acellular capillaries (3.2-Fold) in WT retinas, which was partially ameliorated in p75NTR-/- retinas. GFP-MSCs were successfully incorporated and engrafted into retinal vasculature 1 week post injection and normalized the number of acellular capillaries in p75NTR-/- retinas, yet ischemic WT retinas maintained a 2-Fold increase. Silencing p75NTR on GFP-MSCs coincided with a higher number of cells homing to the ischemic WT retinal vasculature and normalized the number of acellular capillaries when compared to ischemic WT retinas receiving scrambled-GFP-MSCs. In vitro, silencing p75NTR-MSCs enhanced their secretome, as evidenced by significant increases in SDF-1, VEGF and NGF release in MSCs conditioned medium; improved paracrine angiogenic response in HREs, where HREs showed enhanced migration (1.4-Fold) and tube formation (2-Fold) compared to controls. In parallel, modulating MSCs-p75NTR using LM11A-31 resulted in a similar improvement in MSCs secretome and the enhanced paracrine angiogenic potential of HREs. Further, intervention with LM11A-31 significantly mitigated the decline in visual acuity post retinal I/R injury. In conclusion, p75NTR modulation can potentiate the therapeutic potential of MSCs to harness vascular repair in ischemic retinopathy diseases.

Long Non-coding RNA MALAT1 Is Depleted With Age in Skeletal Muscle in vivo and MALAT1 Silencing Increases Expression of TGF-ß1 in vitro.

Long non-coding RNAs (lncRNAs) are thought to function as "sponges" for microRNAs, but a role for such competing endogenous RNAs (ceRNAs) in muscle aging is not well understood. We therefore examined in skeletal muscles of young (4-6 months) and aged (22-24) male and female mice the expression of lncRNA MALAT1, which is predicted in silico to bind the senescence-associated microRNA miR-34a-5p. Results indicate a significant decrease in lncRNA MALAT1 expression in mouse skeletal muscle with age that coincides with an age-related increase in miR-34a-5p expression. In vitro studies using mouse C2C12 myoblasts demonstrate that MALAT1 silencing using siRNA increases miR-34a expression, consistent with a role for MALAT1 as an inhibitor of miR-34a-5p activity. Levels of reactive oxygen species (ROS) are known to increase in muscle with age, and so we treated C2C12 cells with hydrogen peroxide (10 and 100 µM) to examine changes in MALAT1 expression. MALAT1 expression decreased significantly with H2O2 treatment, but this effect was attenuated with p53 siRNA. Finally, miR-34a-5p is implicated in tissue fibrosis, and so we assessed the expression of TGF-ß1 after MALAT1 silencing. MALAT1 siRNA significantly increased the expression of TGF-ß1 in C2C12 cells. These findings suggest that age-related fibrosis and muscle atrophy mediated by ROS may result at least in part from an increase in miR-34a bioavailability resulting from a decline in miR-34a "sponging" due to ceRNA MALAT1 depletion. Crosstalk between MALAT1 and miR-34a may therefore represent a therapeutic target for improving muscle function with aging.

MicroRNAs are critical regulators of senescence and aging in mesenchymal stem cells.

MicroRNAs (miRNAs) have recently come under scrutiny for their role in various age-related diseases. Similarly, cellular senescence has been linked to disease and aging. MicroRNAs and senescence likely play an intertwined role in driving these pathologic states. In this review, we present the connection between these two drivers of age-related disease concerning mesenchymal stem cells (MSCs). First, we summarize key miRNAs that are differentially expressed in MSCs and other musculoskeletal lineage cells during senescence and aging. Additionally, we also reviewed miRNAs that are regulated via traditional senescence-associated secretory phenotype (SASP) cytokines in MSC. Lastly, we summarize miRNAs that have been found to target components of the cell cycle arrest pathways inherently activated in senescence. This review attempts to highlight potential miRNA targets for regenerative medicine applications in age-related musculoskeletal disease.

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.

Kynurenine suppresses osteoblastic cell energetics in vitro and osteoblast numbers in vivo.

Aging is a progressive process associated with declining tissue function over time. Kynurenine, an oxidized metabolite of the essential amino acid tryptophan that increases in abundance with age, drives cellular processes of aging and dysfunction in many tissues, and recent work has focused on understanding the pathways involved in the harmful effects of kynurenine on bone. In this study, we sought to investigate the effects of controlled kynurenine administration on osteoblast bioenergetics, in vivo osteoblast abundance, and marrow fat accumulation. Additionally, as an extension of earlier studies with dietary administration of kynurenine, we investigated the effects of kynurenine on Hdac3 and NCoR1 expression and enzymatic deacetylase activity as potential mechanistic contributors to the effects of kynurenine on osteoblasts. Kynurenine administration suppressed cellular metabolism in osteoblasts at least in part through impaired mitochondrial respiration, and suppressed osteoblastic numbers in vivo with no concurrent effects on marrow adiposity. Deleterious effects of kynurenine treatment on osteoblasts were more pronounced in female models as compared to males. However, kynurenine treatment did not inhibit Hdac3's enzymatic deacetylase activity nor its repression of downstream glucocorticoid signaling. As such, future work will be necessary to determine the mechanisms by which increased kynurenine contributes to aging bone bioenergetics. The current study provides novel further support for the idea that kynurenine contributes to impaired osteoblastic function, and suggests that impaired matrix production by kynurenine-affected osteoblasts is attributed in part to impaired osteoblastic bioenergetics. As circulating kynurenine levels in increase with age, and human bone density inversely correlates with the serum kynurenine to tryptophan ratio, these mechanisms may have important relevance in the etiology and pathogenesis of osteoporosis in humans.