Cheng- Hu

Tumor necrosis factor α suppresses the mesenchymal stem cell osteogenesis promoter miR-21 in estrogen deficiency–induced osteoporosis

Inflammatory cytokines, especially tumor necrosis factor α (TNF‐α), have been shown to inhibit osteogenic differentiation of mesenchymal stem cells (MSCs) and bone formation in estrogen deficiency–induced osteoporosis, but the mechanism responsible remains poorly understood. MicroRNAs (miRNAs) have been shown to regulate MSC differentiation. Here, we identified a novel mechanism whereby TNF‐α, suppressing the functional axis of a key miRNA (miR‐21) contributes to estrogen deficiency–induced osteoporosis. In this study, we screened differentially expressed miRNAs in MSCs derived from estrogen deficiency‐induced osteoporosis and found miR‐21 was significantly downregulated. miR‐21 was suppressed by TNF‐α during the osteogenesis of MSCs. Furthermore, miR‐21 was confirmed to promote the osteoblast differentiation of MSCs by repressing Spry1, which can negatively regulate the osteogenic differentiation of MSCs. Upregulating miR‐21 partially rescued TNF‐α–impaired osteogenesis of MSCs. Blocking TNF‐α ameliorated the inflammatory environment and significantly enhanced bone formation with increased miR‐21 expression and suppressed Spry1 expression in ovariectomized (OVX) mice. Our results revealed a novel function for miR‐21 and suggested that suppressed miR‐21 may contribute to impaired bone formation by elevated TNF‐α in estrogen deficiency–induced osteoporosis. This study may indicate a molecular basis for novel therapeutic strategies against osteoporosis and other inflammatory bone diseases.

MiR-17 modulates osteogenic differentiation through a coherent feed-forward loop in mesenchymal stem cells isolated from periodontal ligaments of patients with periodontitis.

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis, are the most common causes of bone tissue destruction. Recently, human periodontal ligament tissue‐derived mesenchymal stem cells (PDLSCs), a population of multipotent stem cells, have been used to reconstruct tissues destroyed by chronic inflammation. However, the impact of the local inflammatory microenvironment on tissue‐specific stem cells and the mechanisms controlling the effects of the local inflammatory environment remain poorly understood. In this study, we found that the multidifferentiation potential of mesenchymal stem cells (MSCs) isolated from periodontitis‐affected periodontal ligament tissue (P‐PDLSCs) was significantly lower than that of MSCs isolated from healthy human periodontal ligament tissue (H‐PDLSCs). Inflammation in the microenvironment resulted in an inhibition of miR‐17 levels, and a perturbation in the expression of miR‐17 partly reversed the differentiation potential of PDLSCs in this microenvironment. Furthermore, inflammation in the microenvironment promoted the expression of Smad ubiquitin regulatory factor one (Smurf1), an important negative regulator of MSC osteogenic differentiation. Western blotting and 3′ untranslated regions (3′‐UTR) reporter assays confirmed that Smurf1 is a direct target of miR‐17 in PDLSCs. Our data demonstrate that excessive inflammatory cytokine levels, miR‐17, and Smurf1 were all involved in a coherent feed‐forward loop. In this circuit, inflammatory cytokines led to direct activation of Smurf1 and downregulation of miR‐17, thereby increasing degradation of Smurf1‐mediated osteoblast‐specific factors. The elucidation of the molecular mechanisms governing MSC osteogenic differentiation in a chronic inflammatory microenvironment could provide us with a better knowledge of chronic inflammatory disorder and improve stem cell‐mediated inflammatory bone disease therapy. STEM CELLS 2011;29:1804–1816

Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor κB pathway

IntroductionPeriodontitis is initiated and sustained by bacteria. However, the mechanism of bacteria induced periodontitis is still unknown. We hypothesized that bacterial components can affect the functions of stem cells in the periodontium. In this study, we comparatively investigated the influence of Lipopolysaccharide (LPS) on the osteogenesis potential of human periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMMSCs).MethodsHuman PDLSCs and BMMSCs were harvested and mineralized nodule formation was assessed by alizarin red S staining. Expression level of osteogenic related gene was detected by quantitative RT-PCR (qRT-PCR). The expression of Toll-like receptor 4 (TLR4) and its downstream signaling pathway were examined by western blot. The role of TLR4 and related signaling pathway in LPS impairing the osteogenic potential of human PDLSCs and BMMSCs were also studied by alizarin red S staining and qRT-PCR. Experimental periodontitis was induced in adult Sprague–Dawley rats and the alveolar bone loss was measured by micro computed tomography analysis. The expression of alkaline phosphatase (ALP) was assessed by immunohistochemistry and the number of osteoclasts was shown by Tartrate-resistant acid phosphatase (TRAP) staining.ResultsLPS decreased the osteogenic differentiation of human PDLSCs through TLR4 regulated nuclear factor (NF)-κB pathway, but not for BMMSCs. Blocking TLR4 or NF-κB signaling partially reversed the decreased osteogenic potential of PDLSCs and prevented the alveolar bone loss caused by LPS experimental periodontitis in rats. The ALP expression in the periodontal ligament was elevated after treatment with anti-TLR4 antibody or pyrrolidinedithiocarbamate, whereas there was no statistical significance among groups for the number of osteoclasts.ConclusionsThese data suggest that LPS can activate TLR4 regulated NF-κB pathway of human PDLSCs, thus decreasing their osteogenic potential. Blockage of TLR4 or NF-κB pathway might provide a new approach for periodontitis treatment.

GSK3β is a checkpoint for TNF-α-mediated impaired osteogenic differentiation of mesenchymal stem cells in inflammatory microenvironments.

BACKGROUND The fate and differentiation of mesenchymal stem cells (MSCs) depend on various microenvironmental cues. In chronic inflammatory bone disease, bone regeneration is inhibited. The present study therefore sought to identify the underlying molecule mechanisms. METHODS We isolated periodontal ligament stem cells (PDLSCs), a new population of MSCs, from the periodontal ligament tissues of periodontitis patients and healthy controls (p-PDLSCs and h-PDLSCs). The secretion of inflammatory cytokines, like TNF-α, IL-1β, IL-6 and IL-8, after LPS stimulation was measured by ELISA. The expressions of p-GSK3β and GSK3β in two types of PDLSCs were detected by Western blot. TOPFlash was used to assay the Tcf/Lef transcriptional activity. Knockdown of GSK3β by siRNA and over-expression of GSK3β by adenoviruses were performed to confirm the role of GSK3β in the impaired osteogenic differentiation of PDLSCs under inflammatory microenvironment. RESULTS We demonstrated that p-PDLSCs displayed impaired osteogenic capacity than h-PDLSCs. Upon inflammatory stimulation, monocytes, but not PDLSCs, released inflammatory cytokines among which TNF-α directly act on PDLSCs and suppressed their osteogenic differentiation. TNF-α induced the phosphorylation of GSK3β, the deactivated form of GSK3β, which increased nuclear β-catenin and Lef-1 accumulation, and eventually reduced the Runx2-associated osteogenesis in PDLSCs. Over-expression of GSK3β rescued osteogenesis in TNF-α-stimulated PDLSCs, whereas inactivation of GSK3β was sufficient to liberate the β-catenin/Lef-1/Runx2 pathway. CONCLUSION GSK3β plays an obligatory role in the TNF-α-mediated inhibition of osteogenesis in MSCs. GENERAL SIGNIFICANCE The strategy to target GSK3β may provide a potential approach to bone regeneration in inflammatory microenvironments.

TNF-α Inhibits FoxO1 by Upregulating miR-705 to Aggravate Oxidative Damage in Bone Marrow-Derived Mesenchymal Stem Cells during Osteoporosis

Decline of antioxidant defense after estrogen deficiency leads to oxidative damage in bone marrow‐derived mesenchymal stem cells (BMMSCs), resulting a defect of bone formation in osteoporosis. Forkhead box O1 (FoxO1) protein is crucial for defending physiological oxidative damage in bone. But whether FoxO1 is involved in the oxidative damage during osteoporosis is largely unknown. In this study, we found that FoxO1 protein accumulation was decreased in BMMSCs of ovariectomized mice. The decrease of FoxO1 resulted in the suppression of manganese superoxide dismutase (Sod2) and catalase (Cat) expression and accumulation of reactive oxygen species (ROS), inhibiting the osteogenic differentiation of BMMSCs. The decline of FoxO1 protein was caused by tumor necrosis factor‐alpha (TNF‐α) accumulated after estrogen deficiency. Mechanistically, TNF‐α activated NF‐κB pathway to promote microRNA‐705 expression, which function as a repressor of FoxO1 through post‐transcriptional regulation. Inhibition of NF‐κB pathway or knockdown of miR‐705 largely prevented the decline of FoxO1‐mediated antioxidant defense caused by TNF‐α and ameliorated the oxidative damage in osteoporotic BMMSCs. Moreover, the accumulated ROS further activated NF‐κB pathway with TNF‐α, which formed a feed‐forward loop to persistently inhibiting FoxO1 protein accumulation in BMMSCs. In conclusion, our study revealed that the decline of FoxO1 is an important etiology factor of osteoporosis and unclosed a novel mechanism of FoxO1 regulation by TNF‐α. These findings suggested a close correlation between inflammation and oxidative stress in stem cell dysfunction during degenerative bone diseases. Stem Cells 2016;34:1054–1067

Mesenchymal progenitors in osteopenias of diverse pathologies: differential characteristics in the common shift from osteoblastogenesis to adipogenesis.

Osteoporosis is caused by pathologic factors such as aging, hormone deficiency or excess, inflammation, and systemic diseases like diabetes. Bone marrow stromal cells (BMSCs), the mesenchymal progenitors for both osteoblasts and adipocytes, are modulated by niche signals. In differential pathologic states, the pathological characteristics of BMSCs to osteoporoses and functional differences are unknown. Here, we detected that trabecular bone loss co-existed with increased marrow adiposity in 6 osteoporotic models, respectively induced by natural aging, accelerated senescence (SAMP6), ovariectomy (OVX), type 1 diabetes (T1D), excessive glucocorticoids (GIOP) and orchidectomy (ORX). Of the ex vivo characteristics of BMSCs, the colony-forming efficiency and the proliferation rate in aging, SAMP6, OVX, GIOP and ORX models decreased. The apoptosis and cellular senescence increased except in T1D, with up-regulation of p53 and p16 expression. The osteogenesis declined except in GIOP, with corresponding down-regulation of Runt-related transcription factor 2 (RUNX2) expression. The adipogenesis increased in 6 osteoporotic models, with corresponding up-regulation of Peroxisome proliferator activated receptor gamma (PPARγ) expression. These findings revealed differential characteristics of BMSCs in a common shift from osteoblastogenesis to adipogenesis among different osteoporoses and between sexes, and provide theoretical basis for the functional modulation of resident BMSCs in the regenerative therapy for osteoporosis.

DKK1 rescues osteogenic differentiation of mesenchymal stem cells isolated from periodontal ligaments of patients with diabetes mellitus induced periodontitis

Multiple studies have shown that diabetes mellitus is an established risk factor for periodontitis. Recently mesenchymal stem cells derived from periodontal ligament (PDLSCs) have been utilized to reconstruct tissues destroyed by chronic inflammation. However, impact of periodontitis with diabetes mellitus on PDLSCs and mechanisms mediating effects of complex microenvironments remain poorly understood. In this study, we found multiple differentiation potential of PDLSCs from chronic periodontitis with diabetes mellitus donors (D-PDLSCs) was damaged significantly. Inhibition of NF-κB signaling could rescue osteogenic potential of PDLSCs from simple chronic periodontitis patients (P-PDLSCs), whereas did not promote D-PDLSCs osteogenesis. In addition, we found expression of DKK1 in D-PDLSCs did not respond to osteogenic signal and decreased osteogenic potential of D-PDLSCs treated with DKK1 could be reversed. To further elucidate different character between P-PDLSCs and D-PDLSCs, we treated PDLSCs with TNF-α and advanced glycation end products (AGEs), and find out AGEs which enhance effect of TNF-α in PDLSCs might mediate special personality of D-PDLSCs. The adverse effect of AGEs in PDLSCs could be reversed when PDLSCs were treated with DKK1. These results suggested DKK1 mediating WNT signaling might be a therapy target to rescue potential of PDLSCs in periodontitis with diabetes mellitus.

Anti‐aging pharmacology in cutaneous wound healing: effects of metformin, resveratrol, and rapamycin by local application

Cutaneous wounds are among the most common soft tissue injuries and are particularly hard to heal in aging. Caloric restriction (CR) is well documented to extend longevity; pharmacologically, profound rejuvenative effects of CR mimetics have been uncovered, especially metformin (MET), resveratrol (RSV), and rapamycin (RAPA). However, locally applied impacts and functional differences of these agents on wound healing remain to be established. Here, we discovered that chronic topical administration of MET and RSV, but not RAPA, accelerated wound healing with improved epidermis, hair follicles, and collagen deposition in young rodents, and MET exerted more profound effects. Furthermore, locally applied MET and RSV improved vascularization of the wound beds, which were attributed to stimulation of adenosine monophosphate‐activated protein kinase (AMPK) pathway, the key mediator of wound healing. Notably, in aged skin, AMPK pathway was inhibited, correlated with impaired vasculature and reduced healing ability. As therapeutic approaches, local treatments of MET and RSV prevented age‐related AMPK suppression and angiogenic inhibition in wound beds. Moreover, in aged rats, rejuvenative effects of topically applied MET and RSV on cell viability of wound beds were confirmed, of which MET showed more prominent anti‐aging effects. We further verified that only MET promoted wound healing and cutaneous integrity in aged skin. These findings clarified differential effects of CR‐based anti‐aging pharmacology in wound healing, identified critical angiogenic and rejuvenative mechanisms through AMPK pathway in both young and aged skin, and unraveled chronic local application of MET as the optimal and promising regenerative agent in treating cutaneous wound defects.

miR-21 deficiency inhibits osteoclast function and prevents bone loss in mice

MicroRNAs emerge as critical post-transcriptional regulators in bone metabolism. We have previously reported in vitro that miR-21 promotes osteogenesis, while studies have also revealed miR-21 as a regulator of osteoclastogenesis and a promoter of osteoclast differentiation in vitro. However, in vivo data are still lacking in identifying skeletal function of miR-21, particularly its effects on osteoporosis. Here, using miR-21 knockout (miR-21−/−) mice, we investigated effects of miR-21 on bone development, bone remodeling and bone loss. Unexpectedly, miR-21−/− mice demonstrated normal skeletal phenotype in development and maintained osteoblastogenesis in vivo. Besides, miR-21−/− mice showed increased receptor activator of nuclear factor κB ligand (RANKL) and decreased osteoprotegerin (OPG) through miR-21 targeting Sprouty 1 (Spry1). Nevertheless, interestingly, miR-21 deficiency promoted trabecular bone mass accrual physiologically. Furthermore, in pathological states, the protection of bone mass was prominent in miR-21−/− mice. These skeletal effects were attributed to inhibition of bone resorption and osteoclast function by miR-21 deficiency through miR-21 targeting programmed cell death 4 (PDCD4), despite the existence of RANKL. As far as we know, this is the first in vivo evidence of a pro-osteoclastic microRNA. Together, these findings clarified function of miR-21 in bone metabolism, particularly uncovering osteo-protective potential of miR-21 inactivation in osteoporosis.

Recipient Glycemic Micro-environments Govern Therapeutic Effects of Mesenchymal Stem Cell Infusion on Osteopenia.

Therapeutic effects of mesenchymal stem cell (MSC) infusion have been revealed in various human disorders, but impacts of diseased micro-environments are only beginning to be noticed. Donor diabetic hyperglycemia is reported to impair therapeutic efficacy of stem cells. However, whether recipient diabetic condition also affects MSC-mediated therapy is unknown. We and others have previously shown that MSC infusion could cure osteopenia, particularly in ovariectomized (OVX) mice. Here, we discovered impaired MSC therapeutic effects on osteopenia in recipient type 1 diabetes (T1D). Through intensive glycemic control by daily insulin treatments, therapeutic effects of MSCs on osteopenia were maintained. Interestingly, by only transiently restoration of recipient euglycemia using single insulin injection, MSC infusion could also rescue T1D-induced osteopenia. Conversely, under recipient hyperglycemia induced by glucose injection in OVX mice, MSC-mediated therapeutic effects on osteopenia were diminished. Mechanistically, recipient hyperglycemic micro-environments reduce anti-inflammatory capacity of MSCs in osteoporotic therapy through suppressing MSC interaction with T cells via the Adenosine monophosphate-activated protein kinase (AMPK) pathway. We further revealed in diabetic micro-environments, double infusion of MSCs ameliorated osteopenia by anti-inflammation, attributed to the first transplanted MSCs which normalized the recipient glucose homeostasis. Collectively, our findings uncover a previously unrecognized role of recipient glycemic conditions controlling MSC-mediated therapy, and unravel that fulfillment of potent therapeutic effects of MSCs requires tight control of recipient micro-environments.