DataSheet_1_Time-restricted feeding reduces monocyte production by controlling hematopoietic stem and progenitor cells in the bone marrow during obesity.docx

Time-restricted feeding (TRF) has emerged as a promising dietary approach in improving metabolic parameters associated with obesity, but its effect on immune cells under obesogenic condition is poorly understood. We conducted this study to determine whether TRF exerts its therapeutic benefit over obesity-induced myeloid cell production by analyzing hematopoietic stem and progenitor cells in bone marrow (BM) and immune cell profile in circulation. Male C57BL/6 mice were fed a low-fat diet (LFD) or high-fat diet (HFD) ad libitum for 6 weeks and later a subgroup of HFD mice was switched to a daily 10 h-TRF schedule for another 6 weeks. Mice on HFD ad libitum for 12 weeks had prominent monocytosis and neutrophilia, associated with expansion of BM myeloid progenitors, such as multipotent progenitors, pre-granulocyte/macrophage progenitors, and granulocyte/macrophage progenitors. TRF intervention in overweight and obese mice diminished these changes to a level similar to those seen in mice fed LFD. While having no effect on BM progenitor cell proliferation, TRF reduced expression of Cebpa, a transcription factor required for myeloid differentiation. These results indicate that TRF intervention may help maintain immune cell homeostasis in BM and circulation during obesity, which may in part contribute to health benefits associated with TRF.</p

FGF2 Stimulates COUP-TFII Expression via the MEK1/2 Pathway to Inhibit Osteoblast Differentiation in C3H10T1/2 Cells

<div><p>Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor that regulates many key biological processes, including organ development and cell fate determination. Although the biological functions of COUP-TFII have been studied extensively, little is known about what regulates its gene expression, especially the role of inducible extracellular factors in triggering it. Here we report that COUP-TFII expression is regulated specifically by fibroblast growth factor 2 (FGF2), which mediates activation of the MEK1/2 pathway in mesenchymal lineage C3H10T1/2 cells. Although FGF2 treatment increased cell proliferation, the induction of COUP-TFII expression was dispensable. Instead, FGF2-primed cells in which COUP-TFII expression was induced showed a low potential for osteoblast differentiation, as evidenced by decreases in alkaline phosphatase activity and osteogenic marker gene expression. Reducing COUP-TFII by U0126 or siRNA against COUP-TFII prevented the anti-osteogenic effect of FGF2, indicating that COUP-TFII plays a key role in the FGF2-mediated determination of osteoblast differentiation capability. This report is the first to suggest that FGF2 is an extracellular inducer of COUP-TFII expression and may suppress the osteogenic potential of mesenchymal cells by inducing COUP-TFII expression prior to the onset of osteogenic differentiation.</p></div

FGF2 induces COUP-TFII expression in C3H10T1/2 cells.

<p>(A) C3H10T1/2 cells were cultured in 0.1% FBS-containing DMEM for 24 h and then were treated with the indicated amounts of several extracellular factors. After 24 h, the cells were harvested, and the expression of COUP-TFII was analyzed by real-time RT-PCR. Relative COUP-TFII expression was calculated after normalization to β-actin. (B) C3H10T1/2 cells were cultured in 0.1% or 2% FBS-containing DMEM for 24 h with several extracellular factors, as in panel A. Cell lysates were applied to immunoblotting to analyze COUP-TFII protein levels. The level of β-actin was analyzed as a loading control. Numbers below gel images represent the normalized value of relative COUP-TFII levels. (C, E) After the cells were treated with the indicated amounts of FGF2 for 24 h in the same conditions as in panel A, the expression of COUP-TFII was analyzed by conventional RT-PCR analysis (upper panel) and real-time RT-PCR (lower panel), and by immunoblot analysis. (D, F) Cells were incubated with 10 ng/mL of FGF2 for the indicated time period, cell lysates were prepared, and the expression of COUP-TFII was analyzed as in panels C and E. (G) Effects of repeat treatment of FGF2 on COUP-TFII expression. C3H10T1/2 cells were incubated with 10 ng/mL of FGF2 and were then harvested at the indicated time points to undergo immunoblot analysis. The cells were pre-exposed to 10 ng/mL of FGF2 for first 72 h and then re-exposed (indicated as ball-nocks). COUP-TFII expression was analyzed by means of immunoblot analysis. (A-D) Values for the relative expression of the COUP-TFII gene are expressed as the mean ± SEM of a triplicate reaction of one representative experiment. All experiments were repeated three times. Statistical analysis was performed by ANOVA followed by the Tukey post hoc test. (E-G) Immunoblot bands were quantified by densitometry using Science Lab Image Gauge version 3.0 software (Fujifilm), and the ratio of COUP-TFII/β-actin was determined. Data shown are representative of three independent experiments, and the values are expressed as the mean ± SD of three independent experiments. Statistical analysis was performed by ANOVA with the Bonferroni post hoc test. * p<0.01; ** <i>p</i><0.01; *** <i>p</i><0.001 vs. control.</p

COUP-TFII induction by FGF2 priming leads to a reduction in osteodifferentiation potential.

<p>(A, B) Pre-exposure to FGF2 prior to differentiation stimuli inhibits osteoblast differentiation of C3H10T1/2 cells. (A) Cells were treated with 10 ng/mL of FGF2 every other day for 4 days. After removing FGF2-containing media, cells were incubated with osteogenic media (OM) (50 μg/mL of ascorbic acid, 5 mM of β-glycerophosphate, and 100 ng/mL of BMP2). Cells were stained for alkaline phosphatase activity after 5 days of differentiation (left, ALP staining), and were subjected to alizarin red staining after 10 days of differentiation (right, AR staining). The bar graph shows the relative intensity of AR staining. Cells stained with AR were incubated in 10% cetylpyridinium chloride, and staining was quantified at 562 nm. The ratio of OM/GM was determined. (B) After cells were prepared as in panel A, cells were harvested on day 5 for COUP-TFII, ALP, and Osterix, and on day 10 after differentiation for BSP and osteocalcin (Oc). Total RNA was isolated and subjected to real-time RT-PCR. (C-F) Blocking of COUP-TFII induction abolished the anti-osteogenic effect of FGF2 priming. (C) After COUP-TFII–silenced cells were pretreated with FGF2 as in panel A, osteogenic differentiation was induced for 4 days. Alkaline phosphatase activity in the differentiated cells was analyzed by ALP staining. Control, non-FGF2 treated and control siRNAs-transfected cells; FGF2-primed control, FGF2-treated and control siRNA-transfected cell. (D) Osteogenic differentiated cells were harvested and subjected to real-time RT-PCR to analyze expression levels of Osterix (on day 4), ALP (on day 2), BSP and Oc (on day 10). The relative expression levels of COUP-TFII and Runx2 were analyzed on day 0 (that is, before the onset of differentiation). (E) Cells were pretreated with FGF2 as in panel A in the presence or absence of U0126, and the cells then underwent osteogenic differentiation for 4 days. Alkaline phosphatase activity was determined by ALP staining, and magnified images of the differentiated cells are representative of the relevant wells (left). (F) Before the onset of differentiation (day 0), the cells were harvested for analysis of COUP-TFII levels. The relative expression levels of ALP (on day 2), Osterix (on day 4), and Oc (on day 10) were determined. Representative data from three independent experiments are shown. Values for the relative expression of the indicated genes are expressed as the mean ± SEM of triplicate reactions in one representative experiment. Statistical analysis was performed by ANOVA followed by the Tukey post hoc test. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. (G) Working model for the role of overexpressed COUP-TFII in the FGF2-primed mesenchymal cells. FGF2 priming in uncommitted mesenchymal cells induces COUP-TFII expression via the MEK1/2 pathway and it might bring about low osteogenic potential and high pluripotency.</p

FGF2-induced COUP-TFII expression mediates the MEK1/2 signaling pathway.

<p>(A, B) C3H10T1/2 cells were pretreated with DMSO, 5 μM of U0126, 10 μM of PD98059 (PD), 5 μM of LY294002 (LY), 10 μM of SP600125 (SP), or 10 μM of SB202190 (SB) for 30 min, and then FGF2 was added at a concentration of 10 ng/mL. After 24 h, the expression of COUP-TFII was analyzed by means of real-time RT-PCR (A) and immunoblot analysis (B). Relative COUP-TFII expression was calculated after normalization to β-actin (A). (C, D) FGF2 induction of COUP-TFII expression was abolished by U0126 but not by PD98059. Cells were pretreated with the indicated amounts of U0126 or PD98059 for 30 min, and then FGF2 was added. After 24 h, cell lysates were prepared, and COUP-TFII expression was analyzed by real-time RT-PCR and by immunoblot analysis. (E, F) MEK1 and MEK2 can induce COUP-TFII expression. C3H10T1/2 cells were transfected with the indicated siRNAs (E). After 24 h, the cells were incubated with FGF2 for 24 h. Cell lysates were analyzed by immunoblotting for the indicated proteins. Cells transfected with the indicated siRNAs were co-treated with FGF2 and compounds (10 μM of U0126 and 20 μM of PD98059) (F), as in (D). The levels of COUP-TFII and MEK2 were analyzed by immunoblot analysis. (G) Time course effect of FGF2 treatment on COUP-TFII, c-Fos, c-Jun, and Cyclin D1 expression. Cells were treated with FGF2 and then harvested at the indicated time points to analyze COUP-TFII, c-Jun, c-Fos, and Cyclin D1 protein levels by immunoblot analysis. The relative protein levels of the indicated proteins were calculated after normalization to β-actin. (H) Cells were prepared as in (D), and COUP-TFII, c-Jun, c-Fos, and Cyclin D1 protein levels were analyzed by immunoblotting, and their relative level was calculated after normalization to the β-actin level. All immunoblot data shown are representative of three independent experiments, and the values are expressed as the mean ± SD of three independent experiments. Statistical analysis was performed by ANOVA with the Bonferroni post hoc test. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001 vs. control, # <i>p</i><0.05; ## <i>p</i><0.01; ### <i>p</i><0.001 vs. indicated group.</p

The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway

<div><p>Epidermal growth factor-like repeats and discoidin I-like domain 3 (Edil3) is an extracellular matrix protein containing an Arg-Gly-Asp (RGD) motif that binds integrin. Recently, Edil3 has been implicated in various biological processes, including angiogenesis and cellular differentiation. It can inhibit inflammatory bone destruction. The objective of this study was to explore the role of Edil3 in osteoblast differentiation and its underlying molecular mechanisms. In wild-type mice, high expression levels of Edil3 mRNA were observed in isolated calvaria and tibia/femur bones. Immunohistochemical analysis showed that Edil3 protein was localized along periosteum and calcified regions surrounding bone tissues. When murine calvaria-derived MC3T3-E1 cells were cultured in osteogenic medium containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate, Edil3 mRNA and protein expression levels were increased. Treatment with Edil3 protein in growth media increased expression levels of alkaline phosphatase and osteocalcin gene and phosphorylation level of extracellular signal-regulated kinase (ERK). Edil3 treatment with osteogenic medium induced mineralization. Treatment with a neutralizing antibody against α5β1 and MEK inhibitor U0126 inhibited Edil3-enhanced osteogenic marker gene expression and mineral deposition. Edil3 increased protein expression levels of transcription factor runt-related transcription factor2 (Runx2). Edil3-induced Runx2 protein expression was suppressed by pretreatment with U0126. Taken together, these results suggest that Edil3 may stimulate osteoblast differentiation and matrix mineralization by increasing expression of Runx2 through α5β1 integrin /ERK pathway.</p></div

Effects of Edil3 treatment on Runx2 expression and transcriptional activity.

<p>(A) Runx2 protein expression. MC3T3-E1 cells were treated with or without 200 ng/ml of Edil3 for indicated time periods and then subjected to Western blot analysis. Upper panel is a representative image. Lower graph shows quantitative and relative level of Runx2 protein (n = 3). *, <i>P < 0</i>.<i>05</i>; **, <i>P < 0</i>.<i>01</i> vs. before treatment. ^#, <i>P < 0</i>.<i>05</i>; ^##, <i>P < 0</i>.<i>01</i> vs. time control group. (B) Cells were cultured with specific inhibitors (MEK/ERK inhibitor U0126, 10 μM; p38 inhibitor SB203580, 10 μM; or Akt inhibitor LY294002, 10 μM) for 1 h followed by treatment with 200 ng/ml of Edil3 for 4 days. Cells were then harvested for RT-PCR. (C) Cells were treated with Edil3 in the presence or absence of U0126 (+, 5 μM; ++, 10 μM) for 1 h. After changing into growth medium containing 200 ng/ml of Edil3, cells were cultured for 48 h. Western blot analysis was performed with indicated antibodies to evaluate Runx2 expression. Upper panel is a representative image and lower graph shows quantitative level of Runx2 protein (n = 3). *, <i>P < 0</i>.<i>05</i> vs. indicated group. (D) Effects of Edil3 on luciferase activity using a Runx2 luciferase reporter plasmid containing six copies of Runx2-binding osteoblast specific element (6x OSE-Luc). MC3T3-E1 cells were co-transfected with indicated plasmids, including 6x OSE-luc (500 ng), pYX-Asc-Edil3 (+, 300 ng; ++, 600 ng), pCS-Myc-Runx2 (600 ng), and a control mock plasmid (600 ng) with pCMV-β-galactosidase (500 ng). Luciferase activity was measured and normalized to β-galactosidase activity. *, <i>P < 0</i>.<i>05</i>; **, <i>P < 0</i>.<i>01</i> vs. Mock control.</p

Effects of Edil3 protein treatment on osteoblast differentiation.

<p>(A, B) MC3T3-E1 cells were treated for indicated days with 200 ng/ml of Edil3 protein in the presence or absence of osteogenic medium. Cells were then harvested and RT-PCR (left panel) and qRT-PCR (middle and right panels) were performed using specific primers. The number indicates fold ratio between both groups. *, <i>P < 0</i>.<i>05;</i> **, <i>P < 0</i>.<i>01</i>. (C) Cells were treated with indicated concentration of Edil3 protein in growth medium (GM). After 4 days of treatment, cells were harvested and RT-PCR was performed. (D) Cells were cultured with indicated concentrations of Edil3 protein for 14 days in the presence of osteogenic medium and stained with alizarin red solution. (E) Graph showing quantified staining levels in (D). *, <i>P < 0</i>.<i>05</i> compared to OM-treated group.</p

Expression of Edil3 mRNA and protein during osteoblast differentiation.

<p>(A) Endogenous expression of Edil3 mRNA in various progenitor cells. Total RNAs were isolated from cells that had been cultured for 3 days and used for RT-PCR with mouse Edil3 and β–actin primers. (B) Expression of Edil3 mRNA during BMP2-induced osteoblast differentiation. MC3T3-E1 cells were cultured with BMP2 (200 ng/ml) for up to 8 days. (C, D) MC3T3-E1 cells were maintained for up to 6 days in osteogenic medium (OM: α-MEM containing 10% FBS, 50 μg/ml ascorbic acid, and 5 mM β-glycerophosphate) and harvested at the indicated time points. RT-PCR was carried out using specific primers after total RNA isolation (B, C). Western blot analysis was performed with Edil3 and β-actin antibodies to evaluate Edil3 protein expression (D).</p

Effects of Edil3 on osteoblast differentiation following treatment with integrin antibodies.

<p>(A) Integrin expression in MC3T3-E1 cells. Flow cytometry analysis revealed that the distribution of cells stained for integrins (shaded regions) and IgG as background control (open regions). (B) Effects of integrin-blocking antibodies on Edil3-induced osteoblast differentiation. Cells were incubated with 1 μg/ml of specific or control antibodies and 200 ng/ml of Edil3 for 2 days, and then harvested for RT-PCR analysis. (C) MC3T3-E1 cells were treated with Edil3 and indicated antibodies against integrins, and maintained for 14 days in osteogenic medium. These cells were stained with alizarin red solution. (D) Graph showing quantification of staining levels in (C). **, <i>P < 0</i>.<i>01</i> vs. indicated group. mIgG, mouse IgG; rIgG, rat IgG.</p