The impact of ischemic stroke on bone marrow microenvironment and extracellular vesicles: A study on inflammatory and molecular changes.

An ischemic stroke (IS) is caused due to the lack of blood flow to cerebral tissue. Most of the studies have focused on how stroke affects the localized tissue, but it has been observed that a stroke can cause secondary complications in distant organs, such as Bone Marrow (BM). Our study focused on the effect of ischemic strokes on the bone marrow microenvironment. Bone marrow (BM) is a vital organ that maintains inflammatory homeostasis and aids in the repair of damaged tissue after injury/IS. We used the middle cerebral artery occlusion (MCAO) model of ischemic stroke on adult mice (6 months) and investigated the changes in the BM environment. BM cells were used for western blot and RT-PCR, and the BM supernatant was used for cytokine analysis and extracellular vesicle (EVs) isolation. We observed a significant increase in the total cell number within the BM and an increase in TNF-alpha and MCP-1, which are known for inducing a pro-inflammatory environment. Western blots analysis on the whole BM cell lysate demonstrated elevated levels of inflammatory factors (IL-6, TNF-alpha, and TLR-4) and senescence markers (p21 p16). EVs isolated from the BM supernatant showed no change in size or concentration; however, we found that the EVs carried increased miRNA-141-3p and miRNA-34a. Proteomic analysis on BM-derived EVs showed an alteration in the protein cargo of IS. We observed an increase in FgB, C3, Fn1, and Tra2b levels. The signaling pathway analysis showed mitochondrial function is most affected within the bone marrow. Our study demonstrated that IS induces changes in the BM environment and EVs secreted in the BM.

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.

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.

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.

Sex Differences in Adipose Tissue Distribution Determine Susceptibility to Neuroinflammation in Mice With Dietary Obesity.

Preferential energy storage in subcutaneous adipose tissue (SAT) confers protection against obesity-induced pathophysiology in females. Females also exhibit distinct immunological responses, relative to males. These differences are often attributed to sex hormones, but reciprocal interactions between metabolism, immunity, and gonadal steroids remain poorly understood. We systematically characterized adipose tissue hypertrophy, sex steroids, and inflammation in male and female mice after increasing durations of high-fat diet (HFD)-induced obesity. After observing that sex differences in adipose tissue distribution before HFD were correlated with lasting protection against inflammation in females, we hypothesized that a priori differences in the ratio of subcutaneous to visceral fat might mediate this relationship. To test this, male and female mice underwent SAT lipectomy (LPX) or sham surgery before HFD challenge, followed by analysis of glial reactivity, adipose tissue inflammation, and reproductive steroids. Because LPX eliminated female resistance to the proinflammatory effects of HFD without changing circulating sex hormones, we conclude that sexually dimorphic organization of subcutaneous and visceral fat determines susceptibility to inflammation in obesity.

Update on the Role of Glucocorticoid Signaling in Osteoblasts and Bone Marrow Adipocytes During Aging.

PURPOSE OF REVIEW: Bone marrow adipose tissue (BMAT) in the skeleton likely plays a variety of physiological and pathophysiological roles that are not yet fully understood. In elucidating the complex relationship between bone and BMAT, glucocorticoids (GCs) are positioned to play a key role, as they have been implicated in the differentiation of bone marrow mesenchymal stem cells (BMSCs) between osteogenic and adipogenic lineages. The purpose of this review is to illuminate aspects of both endogenous and exogenous GC signaling, including the influence of GC receptors, in mechanisms of bone aging including relationships to BMAT. RECENT FINDINGS: Harmful effects of GCs on bone mass involve several cellular pathways and events that can include BMSC differentiation bias toward adipogenesis and the influence of mature BMAT on bone remodeling through crosstalk. Interestingly, BMAT involvement remains poorly explored in GC-induced osteoporosis and warrants further investigation. This review provides an update on the current understanding of the role of glucocorticoids in the biology of osteoblasts and bone marrow adipocytes (BMAds).

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).

Nanostring-Based Identification of the Gene Expression Profile in Trigger Finger Samples.

Trigger finger is a common yet vastly understudied fibroproliferative hand pathology, severely affecting patients' quality of life. Consistent trauma due to inadequate positioning within the afflicted finger's tendon/pulley system leads to cellular dysregulation and eventual fibrosis. While the genetic characteristics of the fibrotic tissue in the trigger finger have been studied, the pathways that govern the initiation and propagation of fibrosis are still unknown. The complete gene expression profile of the trigger finger has never been explored. Our study has used the Nanostring nCounter gene expression assay to investigate the molecular signaling involved in trigger finger pathogenesis. We collected samples from patients undergoing trigger finger (n = 4) release surgery and compared the gene expression to carpal tunnel tissue (n = 4). Nanostring nCounter analysis identified 165 genes that were differentially regulated; 145 of these genes were upregulated, whereas 20 genes were downregulated. We found that several collagen genes were significantly upregulated, and a regulatory matrix metalloproteinase (MMP), MMP-3, was downregulated. Bioinformatic analysis revealed that several known signaling pathways were dysregulated, such as the TGF-ß1 and Wnt signaling pathways. We also found several novel signaling pathways (e.g., PI3K, MAPK, JAK-STAT, and Notch) differentially regulated in trigger finger. The outcome of our study helps in understanding the molecular signaling pathway involved in the pathogenesis of the trigger finger.

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.

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.

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.

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.

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.

Accumulation of kynurenine elevates oxidative stress and alters microRNA profile in human bone marrow stromal cells.

Kynurenine, a metabolite of tryptophan breakdown, has been shown to increase with age, and plays a vital role in a number of age-related pathophysiological changes, including bone loss. Accumulation of kynurenine in bone marrow stromal cells (BMSCs) has been associated with a decrease in cell proliferation and differentiation, though the exact mechanism by which kynurenine mediates these changes is poorly understood. MiRNAs have been shown to regulate BMSC function, and accumulation of kynurenine may alter the miRNA expression profile of BMSCs. The aim of this study was to identify differentially expressed miRNAs in human BMSCs in response to treatment with kynurenine, and correlate miRNAs function in BMSCs biology through bioinformatics analysis. Human BMSCs were cultured and treated with and without kynurenine, and subsequent miRNA isolation was performed. MiRNA array was performed to identify differentially expressed miRNA. Microarray analysis identified 50 up-regulated, and 36 down-regulated miRNAs in kynurenine-treated BMSC cultures. Differentially expressed miRNA included miR-1281, miR-330-3p, let-7f-5p, and miR-493-5p, which are important for BMSC proliferation and differentiation. KEGG analysis found up-regulated miRNA targeting glutathione metabolism, a pathway critical for removing oxidative species. Our data support that the kynurenine dependent degenerative effect is partially due to changes in the miRNA profile of BMSCs.

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.