Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells

Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts.Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay.Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis.     

CTNNB1 signaling in sertoli cells downregulates spermatogonial stem cell activity via WNT4

Constitutive activation of the WNT signaling effector CTNNB1 (β-catenin) in the Sertoli cells of the Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) and wild-type mice showed that SSC activity is lost in Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development.     

DDIT3/CHOP promotes autophagy in chondrocytes via SIRT1-AKT pathway

Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.     

LncRNA TUG regulates osteogenic differentiation of bone marrow mesenchymal stem cells via miRNA-204/SIRT 1

Objective:To explore the regulation of LncRNA TUG /miRNA-204/SIRT1 pathway on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), so as to provide a new theoretical basis for the clinical treatment of osteoporosis.Methods:Detect changes of LncRNA and miRNA expression predicted in post-differentiation BMSCs with Western blot and qPCR tests. Verify the regulatory relationship between LncRNA and miRNA, miRNA and SIRT1 through the luciferase reporter assay. Transfect recombinant plasmids with LncRNA and their shRNA or transfected miRNA mimics and inhibitors.Results:According to the bioinformatic prediction, LncRNA TUG/miR-204 affected the regulation of SIRT1 on osteogenic differentiation of BMSCs, which were consistent with the results of luciferase reporter assay, namely, there are direct regulation targets between LncRNA TUG and miR-204, miR-204 and SIRT1. Overexpression and knockdown experiments revealed that LncRNA TUG overexpression/knockdown down/up-regulated miR-204 expression, which otherwise increased/decreased SIRT1 levels, and was positively correlated with osteogenic differentiation of BMSCs. Conversely, miR-204 was negatively correlated with LncRNA TUG and SIRT1, and negatively regulated osteogenic differentiation.Conclusion:This study found the direct regulatory relationship of LncRNA TUG/miR-204/SIRT1 during the osteogenic differentiation of BMSCs, and revealed that SIRT1 positively regulates the osteogenic differentiation of BMSCs, which provides a theoretical basis and potential therapeutic targets for a series of osteogenic differentiation-related diseases including osteoporosis.     

Egr-1 upregulates OPN through direct binding to its promoter and OPN upregulates Egr-1 via the ERK pathway

Early growth response factor-1 (Egr-1) and osteopontin (OPN) play important roles in the migration and proliferation of vascular smooth muscle cells (VSMC), but little is known about their relationship. Therefore, we transfected VSMCs with either Egr-1 cDNA, Opn cDNA, a DNA enzyme designed to target Egr-1 (ED5), or antisense Opn oligodeoxynucleotides and examined changes in Egr-1 and OPN expression at the mRNA and protein levels. OPN expression levels were increased in cells that were stably transfected with Egr-1 cDNA. By contrast, both Egr-1 and OPN expression were reduced when ED5 was transfected into Egr-1-expressing cells. Similarly, Opn transfection upregulated Egr-1 levels, while Opn anti-sense oligodeoxynucleotide transfection decreased Egr-1 expression. ChIP analysis showed that Egr-1 binds to the Opn gene promoter. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor PD98059 inhibited the upregulation of Egr-1 by OPN. We find that Egr-1 and OPN positively regulate each other in VSMCs.     

Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway.

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Glycogen accumulation in adipocyte precursors from elderly and obese subjects triggers inflammation via SIRT1/6 signaling

Dysfunctional adipocyte precursors have emerged as key determinants for obesity‐ and aging‐related inflammation, but the mechanistic basis remains poorly understood. Here, we explored the dysfunctional adipose tissue of elderly and obese individuals focusing on the metabolic and inflammatory state of human adipose‐derived mesenchymal stromal cells (hASCs), and on sirtuins, which link metabolism and inflammation. Both obesity and aging impaired the differentiation potential of hASCs but had a different impact on their proliferative capacity. hASCs from elderly individuals (≥65 years) showed an upregulation of glycolysis‐related genes, which was accompanied by increased lactate secretion and glycogen storage, a phenotype that was exaggerated by obesity. Multiplex protein profiling revealed that the metabolic switch to glycogenesis was associated with a pro‐inflammatory secretome concomitant with a decrease in the protein expression of SIRT1 and SIRT6. siRNA‐mediated knockdown of SIRT1 and SIRT6 in hASCs from lean adults increased the expression of pro‐inflammatory and glycolysis‐related markers, and enforced glycogen deposition by overexpression of protein targeting to glycogen (PTG) led to a downregulation of SIRT1/6 protein levels, mimicking the inflammatory state of hASCs from elderly subjects. Overall, our data point to a glycogen‐SIRT1/6 signaling axis as a driver of age‐related inflammation in adipocyte precursors.     

Melatonin suppresses senescence‐derived mitochondrial dysfunction in mesenchymal stem cells via the HSPA1L–mitophagy pathway

Mesenchymal stem cells (MSCs) are a popular cell source for stem cell‐based therapy. However, continuous ex vivo expansion to acquire large amounts of MSCs for clinical study induces replicative senescence, causing decreased therapeutic efficacy in MSCs. To address this issue, we investigated the effect of melatonin on replicative senescence in MSCs. In senescent MSCs (late passage), replicative senescence decreased mitophagy by inhibiting mitofission, resulting in the augmentation of mitochondrial dysfunction. Treatment with melatonin rescued replicative senescence by enhancing mitophagy and mitochondrial function through upregulation of heat shock 70 kDa protein 1L (HSPA1L). More specifically, we found that melatonin‐induced HSPA1L binds to cellular prion protein (PrP^C), resulting in the recruitment of PrP^C into the mitochondria. The HSPA1L‐PrP^C complex then binds to COX4IA, which is a mitochondrial complex IV protein, leading to an increase in mitochondrial membrane potential and anti‐oxidant enzyme activity. These protective effects were blocked by knockdown of HSPA1L. In a murine hindlimb ischemia model, melatonin‐treated senescent MSCs enhanced functional recovery by increasing blood flow perfusion, limb salvage, and neovascularization. This study, for the first time, suggests that melatonin protects MSCs against replicative senescence during ex vivo expansion for clinical application via mitochondrial quality control.     

C6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes

Six-Carbon (C6-) volatiles, including the aldehydes trans-2-hexenal, hexanal and cis-3-hexenal, as well as their corresponding alcohols, are produced from damaged or wounded plant tissue as a product of the enzymatic activity of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway. Aerial treatment of Arabidopsis seedlings with 10 microM concentrations of trans-2-hexenal induces several genes known to be involved in the plant's defense response, including phenylpropanoid-related genes as well as genes of the LOX pathway. Genes encoding the pathogenesis-related proteins PR-1 or PR-2, however, were not induced. Trans-2-hexenal induction thus closely mimics the group of genes induced by methyl jasmonate (MeJA), also a LOX-derived volatile. However, unlike MeJA, trans-2-hexenal did not induce hydroxymethylglutaryl-coenzyme A reductase (HMGR) or thionin2-1. The inductive effect seemed to be limited to C6-related volatiles, as C8-, C9- and other related volatiles did not induce LOX mRNA levels. As has been demonstrated for MeJA, trans-2-hexenal quantitatively reduced wild-type seed germination. Trans-2-hexenal also reduced the germination frequency of the MeJA resistant Arabidopsis mutant, jar1-1, supporting the notion that trans-2-hexenal and MeJA are recognized via different mechanisms. In addition, trans-2-hexenal had a moderate inhibitory effect on root length relative to similar concentrations of MeJA and was approximately 10-fold less effective than MeJA at inducing anthocyanin accumulation in Arabidopsis seedlings. These results suggest that C6-volatiles of the LOX pathway act as a wound signal in plants, but result in a moderate plant response relative to MeJA at both the physiological and molecular level.     

Quercetin protects against diabetic encephalopathy via SIRT1/NLRP3 pathway in db/db mice

Epidemiological studies have found that diabetes and cognitive dysfunction are closely related. Quercetin has been certified with the effect on improving diabetes mellitus (DM) and cognitive impairment. However, the effect and related mechanism of quercetin on diabetic encephalopathy (DE) are still ambiguous. In this study, we used the db/db mice (diabetic model) to discover whether quercetin could improve DE through the Sirtuin1/NLRP3 (NOD-, LRR- and pyrin domain-containing 3) pathway. Behavioural results (Morris water maze and new object recognition tests) showed that quercetin (70 mg/kg) improved the learning and memory. Furthermore, quercetin alleviated insulin resistance and the level of fasting blood glucose. Besides, Western blot analysis also showed that quercetin increased the protein expressions of nerve- and synapse-related protein, including postsynapticdensity 93 (PSD93), postsynapticdensity 95 (PSD95), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain of db/db mice. Quercetin also increased the protein expression of SIRT1 and decreased the expression of NLRP3 inflammation-related proteins, including NLRP3, the adaptor protein ASC and cleaved Caspase-1, the pro-inflammatory cytokines IL-1β and IL-18. In conclusion, the present results indicate that the SIRT1/NLRP3 pathway may be a crucial mechanism for the neuroprotective effect of quercetin against DE.     

Postprandial triglyceride-rich lipoproteins promote the adipogenic differentiation of adipose-derived mesenchymal stem cells via the LRP1/caveolin-1/AKT1 pathway

Diet-induced obesity (OB) is usually accompanied by hypertriglyceridemia, which is characterized by the accumulation of triglyceride (TG)-rich lipoprotein (TRL) particles in the circulation. We previously found that postprandial TRL combined with insulin induced the adipogenic differentiation of 3T3-L1 preadipocytes, which may represent a key mechanism underlying obesity. However, the specific mechanism and signaling pathway involved in this process remain to be fully elucidated. In this study, we found that, in the postprandial state, patients with obesity had significantly higher levels of TG and remnant cholesterol (RC) than normal-weight controls. In vitro, we found that postprandial TRL, together with insulin, promoted the adipogenic differentiation of adipose-derived mesenchymal stem cells (AMSCs), as evidenced by the increased expression of lipogenesis-related genes and their protein products, including low-density lipoprotein related protein 1 (LRP1). Besides, caveolin-1 (Cav-1) expression was also significantly upregulated under this condition. Cav-1 and LRP1 were observed to interact, and then led to the activation of the PI3K/AKT1 signaling pathway. Meanwhile, the inhibition of LRP1 or Cav-1 significantly attenuated the adipogenic differentiation of AMSCs and downregulated AKT1 phosphorylation levels. Moreover, treatment with a selective AKT1 inhibitor significantly suppressed postprandial TRL and insulin-induced adipogenesis in AMSCs. Combined, our results demonstrated that, in association with insulin, postprandial TRL can promote the adipogenic differentiation of AMSCs in a manner that is dependent on the LRP1/Cav-1-mediated activation of the PI3K/AKT1 signaling pathway. Our findings indicated that a postprandial increase in TRL content is a critical factor in the pathogenesis of hypertriglyceridemia and diet-induced obesity.     

Dulcitol suppresses proliferation and migration of hepatocellular carcinoma via regulating SIRT1/p53 pathway

BackgroundHepatocellular carcinoma (HCC) spreads further with continuance and increasing incidence due to its high-grade malignancy and metastasis. More effectual strategies on blocking proliferation and metastasis of cancer cells should be studied in HCC. Dulcitol, a natural product extracted from euonymus alatus, was reported that it could induce apoptosis of C6 glioma cells. However, the underlying mechanism of Dulcitol on HCC remains unclea.PurposeIn this study, we aimed to reveal the effect and potential mechanisms of Dulcitol on hepatocellular carcinoma in vitro and in vivo.Study design and methods The cell proliferation and apoptosis were evaluated by MTT, Ki-67 and Hoechst 33258/PI double staining. The migratory and invasive abilities of HepG2 cells were measured by wound-healing and transwell assays. Pathological changes of tumor tissue were observed by HE staining and IHC methods. The expression levels of protein were detected using Western Blot analysis.ResultsThe results showed that Dulcitol inhibited HepG2 cells proliferation by down-regulating the protein expression of SIRT1, Bcl-2, along with up-regulating p53, acetylated-p53 (K382), cleaved-caspase9, cleaved-caspase3, Bax, and cytochrome c in a dose-dependent manner. Furthermore, Dulcitol surpressed the migration and invasion of HepG2 cells through decreasing the levels of MMP-2, uPA and MMP-9 and increasing E-cadherin associated with tumor invasion. In vivo, Dulcitol distinctly inhibited the growth of HepG2 cancer xenograft tumors via inhibiting SIRT1/p53 pathway.ConclusionsOur findings suggested that Dulcitol acted as a SIRT1 inhibitor, inducing apoptosis and inhibiting proliferation, migration and invasion of HepG2 cells and its modulatory mechanism seemed to be associated with regulation of MMPs, SIRT1/p53 pathways.     

GDNF-induced leukemia inhibitory factor can mediate differentiation via the MEK/ERK pathway in pheochromocytoma cells derived from nf1-heterozygous knockout mice

Glial cell line-derived neurotrophic factor (GDNF) can induce neuron-like differentiation of mouse pheochromocytoma (MPC) cell lines derived from mice with a heterozygous knockout mutation of nf1, the murine counterpart of the human gene mutated in neurofibromatosis type 1 (NF1). Here, we show that GDNF-induced differentiation in the MPC 862L cell line is mediated by the MEK/extracellular signal-regulated kinase (ERK) pathway. Neurite outgrowth, increased expression of growth-associated protein 43, and decreased incorporation of bromodeoxyuridine (BrdU) were induced by treatment with GDNF, H-RasV12, or a constitutively active MEK2. GDNF also induces leukemia inhibitory factor (LIF) via the MEK/ERK pathway, and LIF itself can elicit these differentiative changes via a cell-extrinsic autocrine/paracrine pathway. Treatment with anti-LIF neutralizing antibody depleted the differentiative activity of the conditioned medium from cells stimulated for MEK/ERK signaling, while recombinant LIF could induce differentiation in MPC cells, indicating that LIF is the sole factor with differentiative activity. LIF could activate MEK1/2 and STAT3, but LIF-induced differentiation was blocked only by the MEK1/2-specific inhibitor U0126, indicating that the MEK/ERK pathway is necessary for LIF action in MPC cells. Our findings suggest that LIF may be utilized for signaling mediated by GDNF and may be important in the pathobiology of neuroendocrine tumors.