Differential responses of bone to angiotensin II and angiotensin(1-7): beneficial effects of ANG(1-7) on bone with exposure to high glucose
Abstract
Osteoporosis, diabetes, and hypertension are common concurrent chronic disorders. This study aimed to explore the respective effects of angiotensin II (ANG II) and angiotensin(1-7) [ANG(1-7)], active peptides in the renin-angiotensin system, on osteoblasts and osteoclasts under high-glucose level, as well as to investigate the osteo-preservative effects of ANG II type 1 receptor (AT1R) blocker and ANG(1-7) in diabetic spontaneously hypertensive rats (SHR). ANG II and ANG(1-7), respectively, decreased and increased the formation of calcified nodules and alkaline phosphatase activity in MC3T3-E1 cells under high-glucose level, and respectively stimulated and inhibited the number of matured osteoclasts and pit resorptive area in RANKL-induced bone marrow macrophages. Olmesartan and Mas receptor antagonist A779 could abolish those effects. ANG II and ANG(1-7), respectively, downregulated and upregulated the expressions of osteogenesis factors in MC3T3-E1 cells. ANG II promoted the expressions of cathepsin K and MMP9 in RAW 264.7 cells, whereas ANG(1-7) repressed these osteoclastogenesis factors. ANG II rapidly increased the phosphorylation of Akt and p38 in RAW 264.7 cells, whereas ANG(1-7) markedly reduced the phosphorylation of p38 and ERK under high-glucose condition. After treatments of diabetic SHR with valsartan and ANG(1-7), a significant increase in trabecular bone area, bone mineral density, and mechanical strength was only found in the ANG(1-7)-treated group. Treatment with ANG(1-7) significantly suppressed the increase in renin expression and ANG II content in the bone of SHR. Taken together, ANG II/AT1R and ANG(1-7)/Mas distinctly regulated the differentiation and functions of osteoblasts and osteoclasts upon exposure to high-glucose condition. ANG(1-7) could protect SHR from diabetes-induced osteoporosis.
INTRODUCTION
Angiotensin II (ANG II), one of the main components in the renin-angiotensin system (RAS), exerts its biological effects by binding to its receptor ANG II type 1 receptor (AT1R) under physiological and pathological states (1, 2). Besides ANG II, several other peptides are now recognized as biologically vital. Of particular importance is the endogenous heptapeptide angiotensin(1-7) [ANG(1-7)] that has emerged as a new metabolite of the RAS decades ago (3). The physiological actions of ANG(1-7) were initially described as displaying protective effects on the cardiovascular system and the kidney by acting on its receptor Mas (3, 4). There is emerging evidence demonstrating that it has a broad range of effects in different organs and tissues beyond the cardio-renal system (5).
The global diabetes prevalence in 2010 among adults (aged 20–79) was 6.4%, affecting 285 million adults, and it is estimated to increase to 7.7%, affecting 439 million adults by 2030 (6). Chronic diabetes mellitus (DM) adversely affects different parts of the body, such as the bone. Low-bone mineral density (BMD) is consistently observed in type 1 DM (T1DM), whereas BMD in T2DM is usually normal or even slightly elevated compared with an age-matched control population (7). Recent meta-analyses and cohort studies confirmed that T1DM and T2DM were associated with higher fracture risk (8, 9) due to bone fragility, which was common in T1DM and T2DM (10). Among the complex pathophysiological mechanisms underlying bone fragility in DM, the tissue RAS in bone, which had been explored in our early study, might be a critical contributor (11). However, few evidence-based studies on the effects of drugs targeting RAS on bone density of patients with diabetes have been reported.
Our group demonstrated that increase in the activity of skeletal RAS shown by the activation of ANG II signaling was involved in the pathological process of type 1 diabetes-induced osteoporosis (12, 13). After showing the presence of ANG(1-7) in mice tibia (14), others reported that ANG(1-7) played a key role in bone remodeling (15) and ameliorated ovariectomy-induced osteoporosis in rats (16). However, the role of ANG II and ANG(1-7) in bone metabolism associated with hyperglycemia and their effects on osteoblasts and osteoclasts under high-glucose (HG) levels are not fully understood.
The aim of this study was to investigate the effects of ANG II and ANG(1-7) on the functions of osteoblasts and osteoclasts upon high-glucose challenge, and to explore the osteo-preservative effects of AT1R blocker and ANG(1-7) in spontaneously hypertensive rats (SHR) with diabetes induced by streptozotocin (STZ).
MATERIALS AND METHODS
Cell Culture
Murine osteoblastic MC3T3-E1 cells and preosteoclastic RAW 264.7 cells were purchased from ATCC (American Type Culture Collection, Manassas, VA) and routinely cultured in α-Minimum Essential Medium (α-MEM, Biosera, Kansas, MO) supplemented with 10% fetal bovine serum (FBS, Shanghai BioSun Sci&Tech Co., Ltd., China) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Biosera) in a humidified incubator with 95% air and 5% CO2 at 37°C. An osteogenic differentiation medium consisting of α-MEM, 10% FBS, sodium β-glycerophosphate (10 mM), and l-ascorbic acid (50 μg/mL) was used to induce the differentiation of osteoblasts in MC3T3-E1 cell line.
Alkaline Phosphatase Activity Assay and Staining
MC3T3-E1 cells were seeded into 24-well plates with a cell density of 1.2 × 104 cells/well, incubated with differentiation medium in low-glucose (LG, 5.5 mM) and HG (30 mM) levels, and treated with angiotensin II (ANG II, 10−7 M, Peptide Institute, Japan) in the absence or presence of olmesartan (OLM, 10−6 M, Sigma, St. Louis, MO), or angiotensin (1-7) [ANG(1-7) (10−7 M, Peptide Institute) in the absence or presence of its receptor blocker A779 (10−6 M, BACHEM, Torrance, CA) when the cells reached 80% confluence in a well. On treatment for 7 days, cells were harvested and lysed with a passive lysis buffer (Promega, Madison, WI). Alkaline phosphatase (ALP) activity was determined by Wako laboratory assay ALP (Wako, Japan). Absorbance was measured at 405 nm by using a spectrophotometric plate reader (Bio-Rad model 550, Japan). ALP activity was normalized by the protein content detected by Bradford (Bio-Rad, Hercules, CA) protein assay (17).
Expression of Osteogenesis Markers in MC3T3-E1 Cells
MC3T3-E1 cells, after incubation with serum-free medium for 12 h, were treated with vehicle (Control), angiotensin II (ANG II, 100 nM) in the absence or presence of ANG II type 1 receptor blocker olmesartan (OLM, 1 μM), or ANG(1-7) (100 nM) in the absence or presence of Mas receptor antagonist (A779, 1 μM) for 72 h. Protein expression of osteogenic factors such as runt-related transcription factor 2 (RUNX2), type 1 collagen (COL-1), and osteocalcin (OCN) was assessed by immunoblotting.
Bone Nodule Formation Assay
MC3T3-E1 cells were cultured in an osteogenic differentiation medium at both LG and HG levels with the tested drugs, including ANG II (10−7 M) in the absence or presence of OLM (10−6 M), or ANG(1-7) (10−7 M) in the absence or presence of its receptor blocker A779 (10−6 M). On treatment for 21 days, cells were fixed and stained with 1% Alizarin Red S. Images of the stained cells were captured under a light microscope (Olympus, Japan). Briefly, 0.5 M HCl and 5% SDS were added to dissolve the bone nodules. The optical density was measured at 415 nm by using a spectrophotometric plate reader (Bio-Rad).
Bone Marrow Cell Culture
Bone marrow macrophages (BMMs) were isolated from the bilateral femurs and tibias of 3-week-old C57BL/6 male mice. Briefly, bone marrow cells were flushed out from the femurs and tibias, collected into tubes, and washed twice with PBS. After lysing the red blood cells, the mononuclear cell-rich fraction was separated from the marrow cells by density gradient centrifugation at 500 g for 20 min and cultured (105 cells/well in 24-well plate) in α-MEM containing 10% FBS and 1% antibiotics (streptomycin and penicillin).
Tartrate-Resistant Acid Phosphatase Staining
BMMs were cultured into 24-well plates and treated with macrophage colony stimulating factor (M-CSF; 40 ng/mL) and RANKL (100 ng/mL) combined with ANG II (10−7 M) in the absence or presence of OLM (10−6 M), or ANG(1-7) (10−7 M) in the absence or presence of its receptor blocker A779 (10−6 M). After treatment for 7 days, cells were fixed with paraformaldehyde (4%, vol/vol) in PBS for 10 min at room temperature before staining with a tartrate-resistant acid phosphatase (TRAP) kit (Sigma). Matured osteoclasts were identified with TRAP-positive cells showing multi-nuclei (>3). The number of matured osteoclasts was counted and calculated in each view.
Pit Formation
BMMs were seeded onto a Corning Osteo Assay Surface 96-well plate (Life Sciences, Tewksbury, MA) at a density of 5 × 103 cells per well. After attaching to the well bottom for 24 h, cells were treated with M-CSF (40 ng/mL) and RANKL (100 ng/mL) combined with ANG II (10−7 M) in the absence or presence of OLM (10−6 M), or ANG(1-7) (10−7 M) in the absence or presence of its receptor blocker A779 (10−6 M) under both LG and HG condition for 1 wk. The cells were treated with 5% sodium hypochlorite for 5 min and washed with distilled water. The images inside the plate wells were captured with microscopy to identify pit formation and the area of pit resorption was quantified using Image-Pro plus 6.0 software (Media Cybernetics, Silver Spring, MD).
Expression of Osteoclastic Markers and Signaling Proteins in RAW 264.7 Cells
RAW 264.7 cells induced with M-CSF (40 ng/mL) and RANKL (100 ng/mL) were treated with vehicle (Control), ANG II (100 nM) in the absence or presence of olmesartan (OLM, 1 μM), or ANG(1-7) (100 nM) in the absence or presence of A779 (1 μM) for 72 h. Protein expression of resorptive factors including cathepsin K and matrix metalloproteinase-9 (MMP9) was measured by immunoblotting. In addition, the signaling pathways of Akt, p38, ERK, and JNK were assessed after incubation of RANKL-induced RAW 264.7 cells with ANG II (100 nM) or ANG(1-7) (100 nM) for 15 min and 30 min.
Animals and Treatments
Male spontaneously hypertensive rats (SHR) were purchased from Slac Laboratory (Shanghai, China). The animals were housed in environmentally controlled central animal facilities at 22°C, kept under light:dark (12-h:12-h) cycles and fed with a commercial diet and distilled water ad libitum during the experimental period. Diabetes was induced in 3-mo-old SHR by intraperitoneal injection with freshly prepared streptozotocin (STZ; dissolved in 10 mM citrate buffer, pH 4.2), which was given once at a dosage of 60 mg/kg. The SHR that were injected with vehicle served as the nondiabetic control (n = 8 rats). The diabetic SHR were daily treated with saline (ip), or valsartan (5 mg/kg ip), or Angiotensin-(1-7) (500 μg/kg ip). The body weight and fasting blood glucose of the rats were monitored during the experimental period. Four weeks after drug administration, the serum, tibias, and femurs of the rats were harvested for a variety of biochemical, histological, and molecular analyses. The animal study protocol was reviewed and approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine. The methods were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, WA).
Systolic Blood Pressure and Diastolic Blood Pressure
The systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the tail artery of conscious rats were measured with a noninvasive computerized tail-cuff system (ALC-NIBP, Alcott Biotech, China). The SBP and DBP were obtained by averaging 10 measurements.
Serum Chemistries
Calcium (Ca) and phosphorus (P) concentrations in serum were measured by standard colorimetric methods (Stanbio Laboratory, Boerne, TX) using a micro-plate reader (BioTek, Winooski, VT). Serum concentrations of procollagen type I N-propeptide (PINP) and tartrate-resistant acid phosphatase 5b (TRAcP-5b) were measured using commercial ELISA assay kits (IDS, UK).
Histological Staining on Bone
The femurs were fixed in 4% (vol/vol) formaldehyde/PBS (pH 7.2), decalcified in 0.5 M EDTA (pH 8.0), and embedded in paraffin by standard histological procedures. Serial sections of 3 µm were prepared at the distal femoral metaphysis. Hematoxylin and eosin (H&E) staining was performed. Trabecular bone quantity expressed as trabecular bone area over total area (BA/TA) was measured using the OsteoMeasure system (OsteoMetrics Inc., Decatur, GA). Tartrate-resistant acid phosphatase (TRAP) staining was used for the identification of osteoclasts following the manufacturer’s instructions (Sigma). Osteoclast-covered bone surface (OcS/BS) was determined by the OsteoMeasure system. Stained slides were visualized under a microscope (Leica DM 2500).
Micro-CT Analysis
The tibia of each animal was scanned to obtain three-dimensional (3D) images and quantitative parameters of trabecular bone at the proximal metaphysis of tibia. The detection process and the setting of relevant detecting parameters were performed as described previously (11). Briefly, the proximal tibial metaphysis underneath the growth plate was examined on a 1.81-mm slab, corresponding to 173 slices, with a high-resolution micro viva-CT80 system (Scanco Medical, Bassersdorf, Switzerland). Hand-drawn contours were used to isolate the metaphyseal region of interest and trabecular compartments. Trabecular bone micro-architecture was assessed using the µCT Evaluation Program (Image Processing Language v. 5.0 A, Scanco). The 3D parameters, including 1) the mean mineral density of total volume (BMD/TV) and 2) bone volume over total volume (BV/TV), were obtained for the trabecular bone.
Biomechanical Strength
The bone strength was measured on intact femurs using a three-point bending test. Each specimen was placed on two supports spaced 15 mm apart and a load was applied to the bone midway between the supports at a deformation rate of 0.5 mm/min until fracture occurred. Load-deformation curves were recorded during the bending process using a material testing machine (Tinius Olsen, H25KT, Horsham, PA). The ultimate force of the cortical bone was calculated by automated computation.
Western Blotting
The proteins from cells and animal tissues were extracted in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai) and Laemmli buffer (Boston Bioproducts, Worcester, MA), respectively. Before protein extraction from bone tissue, bone marrow was removed by flushing with cold PBS via a syringe. Samples containing 30 µg of protein were separated on 10% SDS-PAGE gel, and transferred to nitrocellulose membranes (Whatman). After saturation with 5% (wt/vol) nonfat dry milk in TBS and 0.1% (wt/vol) Tween 20 (TBST), the membranes were incubated with primary antibodies (Table 1) at dilutions ranging from 1:1,000 to 1:2,000 at 4°C overnight. After three washes with TBST, the membranes were incubated with secondary immunoglobulins conjugated to IRDye 800CW Infrared Dye (LI-COR), including donkey anti-goat IgG and donkey anti-mouse IgG at the dilution of 1:15,000. Blots were visualized by the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE). Signals were densitometrically assessed (Odyssey Application Software v 3.0) and normalized to the β-actin signals to correct for unequal loading using the mouse monoclonal anti-β-actin antibody (Sigma).
| Antibody | Specificity | Cross Reactivity | Manufacturer | Catalog No. |
|---|---|---|---|---|
| RUNX2 | Mouse mAb | M, R, H | abcam | ab76956 |
| Type 1 collagen | Rabbit pAb | M, S, C, H, Pg | abcam | ab34710 |
| Osteocalcin | Rabbit pAb | M, H | abcam | ab93876 |
| Cathepsin K | Rabbit pAb | M, R, H, Z | abcam | ab19027 |
| MMP9 | Rabbit pAb | M, R, Dg, H | abcam | ab38898 |
| Renin (A-1) | Mouse mAb | M, R, H | SCB | sc-137252 |
| Angiotensin (H-12) | Mouse mAb | M, R, H | SCB | sc-374511 |
| Akt (pan) (C67E7) | Rabbit mAb | M, H, R, Mk, Dm | CST | #4691 |
| Phospho-Akt (Ser473) | Rabbit mAb | H, M, R, Hm, M, Dm, Z, B | CST | #4060 |
| P44/42 MAPK (Erk1/2) (137F5) | Rabbit mAb | H, M, R, Hm, Mk, Mi, Dm, Z, B, Dg, Pg, Ce | CST | #4695 |
| Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) | Rabbit mAb | H, M, R, Hm, Mk, Mi, Dm, Z, B, Dg, Pg, Sc | CST | #4370 |
| p38 MAPK (D13E1) | Rabbit mAb | H, M, R, Hm, Mk, B, Pg | CST | #8690 |
| Phospho-p38 MAPK (Thr180/Tyr182) | Rabbit mAb | H, M, R, Mk, Mi, Pg, Sc | CST | #4511 |
| JNK2 (56G8) | Rabbit mAb | H, M, R, Hm, Mk, Mi | CST | #9258 |
| Phospho-SAPK/JNK (Thr183/Tyr185) | Rabbit mAb | H, M, R, Dm, Sc | CST | #4668 |
| β-actin | Mouse mAb | R, H, M, B, Ct, Cp, Rb | Sigma | A2228 |
Statistical Analysis
The data from these in vitro and in vivo experiments were reported as means ± standard error of mean (SEM) for each group. All statistical analyses were performed using PRISM version 4.0 (GraphPad). Inter-group differences were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test as a post test to compare the group means if overall P < 0.05. Differences with P value of less than 0.05 were considered statistically significant.
RESULTS
Effects of ANG II and ANG(1-7) on Calcium Nodule Formation
The effects of ANG II and ANG(1-7) on the mineralization of preosteoblast were determined by Alizarin Red S staining (Fig. 1A) and quantified (Fig. 1B) under low (5.5 mM) and high (30 mM) glucose levels in MC3T3-E1 cells. ANG II and ANG(1-7) significantly (P < 0.001) decreased and increased the formation of calcium nodules, and ANG II type 1 receptor OLM and ANG(1-7) receptor Mas antagonist A779 markedly inhibited these effects (P < 0.001), respectively, under high glucose level. While, the effects of ANG II and ANG(1-7) on the mineralization in MC3T3-E1 cells with exposure to LG level were comparable.
Figure 1.Mineralization formation of MC3T3-E1 cells treated with vehicle (control), angiotensin II (ANG II, 100 nM) in the absence or presence of ANG II type 1 receptor blocker olmesartan (OLM, 1 μM), and angiotensin(1-7) [ANG(1-7), 100 nM] in the absence or presence of ANG(1-7) receptor antagonist (A779, 1 μM). A: representative images of calcified nodule formation. B: quantitative measurement of calcified nodules. Results were obtained from three independent experiments in triplicate and were expressed as means ± SE. ***P < 0.001 vs. Control; ###P < 0.001 vs. ANG II; &&&P < 0.001 vs. ANG(1-7).
Effects of ANG II and ANG(1-7) on Differentiation of Preosteoblast
The effects of ANG II and ANG(1-7) on differentiation of preosteoblast were determined by alkaline phosphatase (ALP) staining (Fig. 2A) and ALP activity test (Fig. 2B) under low and high glucose levels in MC3T3-E1 cells. Consistent with the results of nodule mineralization, OLM and A779 could dramatically (P < 0.05) repress the regulatory effects of ANG II and ANG(1-7) on ALP activity, respectively, under high glucose level, however, ALP activity was comparable among groups when MC3T3-E1 cells were incubated at low glucose level.
Figure 2.Differentiation activity of MC3T3-E1 cells treated with vehicle (control), angiotensin II (ANG II, 100 nM) in the absence or presence of ANG II type 1 receptor blocker olmesartan (OLM, 1 μM), and angiotensin(1-7) [ANG(1-7), 100 nM] in the absence or presence of ANG(1-7) receptor antagonist (A779, 1 μM). Results were obtained from three independent experiments in triplicate and were expressed as means ± SE. *P < 0.05, vs. Control; #P < 0.05, vs. ANG II; &P < 0.05, vs. ANG(1-7).
Effects of ANG II and ANG(1-7) on Osteoclastogenesis and Resorption Pits
To further explore the effects of ANG II and ANG(1-7) on osteoclastogenesis and bone resorption activity, the formation of multinuclear cells (Fig. 3, A and B) and resorption pits (Fig. 3, C and D) was determined using bone marrow macrophages (BMMs) under low and high glucose levels. The number of TRAP-positive stained cells was significantly (Fig. 3B, P < 0.05) increased and decreased by treatment of BMMs with ANG II and ANG(1-7), respectively, and OLM and A779 suppressed the effects of ANG II and ANG(1-7) in a glucose-independent manner (P < 0.05). Similarly, cotreatment of BMMs with ANG II plus OLM (Fig. 3D, P < 0.01) or ANG(1-7) plus A779 (P < 0.05) markedly and glucose-independently diminished the rise or the reduction in the formation of resorption pits induced by ANG II or ANG(1-7).
Figure 3.Osteoclastic formation and osteoclasts-involved pit formation in macrophage colony stimulating factor (M-CSF) and RANKL-induced bone marrow macrophages (BMM) treated with vehicle (control), angiotensin II (ANG II, 100 nM) in the absence or presence of ANG II receptor type 1 blocker olmesartan (OLM, 1 μM), and angiotensin(1-7) [ANG(1-7), 100 nM] in the absence or presence of ANG(1-7) receptor antagonist (A779, 1 μM). A: representative images with positive staining of tartrate-resistant acid phosphatase (TRAP) indicating the formation of matured osteoclasts. B: quantitative measurement of TRAP-positive cells. C: representative images of bone resorption pits. D: quantitative measurement of resorption pit area. Results were obtained from three independent experiments in triplicate and were expressed as means ± SE. *P < 0.05, ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. ANG II; &P < 0.05, &&P < 0.01 vs. ANG(1-7).
Effects of ANG II and ANG(1-7) on Osteoblast-Specific Factors
Protein expression of RUNX2, COL-1, and OCN was assessed in MC3T3-E1 cells after treatment with ANG II and ANG(1-7) in the absence and the presence of respective receptor blocker (Fig. 4A). Immunoblotting analysis showed that ANG II markedly downregulated the expression of osteoblastic transcriptional factor RUNX2 (Fig. 4B, P < 0.05) and osteoblastic markers COL-1 and OCN (P < 0.05) at both low and high glucose levels. The protein expression of RUNX2, COL-1, and OCN was higher (P < 0.05) in the ANG(1-7)-treated group than those in the control group regardless of their glucose level. Treatment with OLM remarkably reversed the action of ANG II on RUNX2, COL-1, and OCN, and A779 obviously blocked the effects of ANG(1-7) on COL-1 and OCN in MC3T3-E1 cells.
Figure 4.Protein expression of osteogenic factors in MC3T3-E1 cells treated with vehicle (control), angiotensin II (ANG II, 100 nM) in the absence or presence of ANG II type 1 receptor blocker olmesartan (OLM, 1 μM), and angiotensin(1-7) [ANG(1-7), 100 nM] in the absence or presence of ANG(1-7) receptor antagonist (A779, 1 μM). A: immunoblotting bands. B: quantitative data for targeted proteins. Results were obtained from three independent experiments in triplicate and were expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; #P < 0.05, ###P < 0.001 vs. ANG II; &P < 0.05, &&&P < 0.001 vs. ANG(1-7). ARB, angiotensin receptor blocker; COL-1, type 1 collagen; OCN, osteocalcin; RUNX2, runt-related transcription factor 2.
Effects of ANG II and ANG(1-7) on Osteoclast-Specific Factors
The effects of ANG II and ANG(1-7) on protein expression of bone resorption factors including cathepsin K and MMP9 were studied in RAW 264.7 cells (Fig. 5A). Treatment with OLM (P < 0.001) and A779 (P < 0.001) significantly suppressed ANG II-induced upregulation and ANG(1-7)-induced downregulation of MMP9 expression at low glucose level (Fig. 5B), respectively, and similar effects were observed in RAW 264.7 cells treated with high glucose level. ANG II could increase (P < 0.001) cathepsin K protein expression at low glucose level and ANG(1-7) could decrease (P < 0.05) cathepsin K protein expression at high glucose level, but neither OLM nor A779 could attenuate those effects.
Figure 5.Protein expression of osteoclastic resorption factors in macrophage colony stimulating factor (M-CSF) and RANKL-induced RAW 264.7 cells treated with vehicle (control), angiotensin II (ANG II, 100 nM) in the absence or presence of ANG II type 1 receptor blocker olmesartan (OLM, 1 μM), and angiotensin(1-7) [ANG(1-7), 100 nM] in the absence or presence of ANG(1-7) receptor antagonist (A779, 1 μM). A: immunoblotting bands. B: quantitative data for targeted proteins. Results were obtained from three independent experiments in triplicate and were expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; ###P < 0.001 vs. ANG II; &&P < 0.01, &&&P < 0.001 vs. ANG(1-7). MMP9, matrix metalloproteinase-9.
Regulation of ANG II on Osteoclastic Signaling Pathway
Mitogen-activated protein kinase (MAPK) signal transduction involves p38, ERK, and JNK, which are the most important signaling molecules known to regulate osteoclastogenesis and osteoclastic activity. The MAPK and Akt signaling pathways were evaluated in RAW 264.7 cells in response to ANG II treatment (Fig. 6A). Treatment with ANG II rapidly increased phosphorylation of Akt and p38 MAPK at 15 min under the medium condition with both low (Fig. 6B, P < 0.001) and high glucose levels (P < 0.05), whereas the activation of the signaling pathways at 30 min was only observed for Akt phosphorylation under low glucose level (P < 0.001). ANG II did not influence RANKL-induced ERK phosphorylation regardless of incubation time and glucose level. In addition, the phosphorylation of JNK was significantly stimulated by ANG II after treatment for 30 min at high glucose level (P < 0.001).
Figure 6.Effects of angiotensin II (ANG II) on signaling pathways involved in osteoclastogenesis under low- and high-glucose levels. RAW 264.7 cells induced with macrophage colony stimulating factor (M-CSF) and RANKL were treated with ANG II (100 nM) for 15 min and 30 min, respectively. Immunoblotting analysis on expression of proteins involved in Akt, p38, ERK, and JNK pathways was performed. A: immunoblotting bands. B: the quantitative results on the ratio of p-Akt/Akt, p-p38/p38, p-ERK/ERK, and p-JNK/JNK. Results were obtained from three independent experiments in triplicate and were expressed as means ± SE. *P < 0.05, ***P < 0.001 vs. RANKL-treated group at 15 min; #P < 0.05, ###P < 0.001 vs. RANKL-treated group at 30 min.
Regulation of ANG(1-7) on Osteoclastic Signaling Pathway
Besides ANG II, we also determined the regulation of ANG(1-7) on osteoclastic signaling pathways including Akt, p38 MAPK, ERK, and JNK in RAW 264.7 cells (Fig. 7A). ANG(1-7) markedly decreased the phosphorylation of p38 MAPK and ERK at both time points (15 min and 30 min) under high glucose level (Fig. 7B, P < 0.001). However, under low glucose level, treatment with ANG(1-7) did not affect phosphorylation of p38 MAPK or ERK, but led to a reduction in phosphorylation of JNK at 15 min (P < 0.01).
Figure 7.Effects of angiotensin(1-7) [ANG(1-7)] on signaling pathways involved in osteoclastogenesis under low- and high-glucose levels. RAW 264.7 cells induced with macrophage colony stimulating factor (M-CSF) and RANKL were treated with ANG(1-7) (100 nM) for 15 min and 30 min, respectively. Immunoblotting analysis on expression of proteins involved in Akt, p38, ERK, and JNK pathways was performed. A: immunoblotting bands. B: the quantitative results on the ratio of p-Akt/Akt, p-p38/p38, p-ERK/ERK, and p-JNK/JNK. Results were obtained from three independent experiments in triplicate and were expressed as means ± SE. **P <0.01, ***P < 0.001 vs. RANKL-treated group at 15 min; ###P < 0.001 vs. RANKL-treated group at 30 min.
Effects of Valsartan and ANG(1-7) on Physiological Parameters of SHR
Hyperglycemia was developed in male spontaneously hypertensive rats (Table 2). Valsartan and ANG(1-7) did not change the increase in the level of fasting blood glucose, or the decrease in body weight (Fig. 8A) of diabetic SHR during the 4 wk of experiment, whereas treatment with valsartan dramatically reduced the SBP (Fig. 8B, P < 0.05) and DBP (Fig. 8C, P < 0.01), both of which were not altered in the ANG(1-7)-treated group as compared with the vehicle-treated group.
Figure 8.Physiological parameters of non-diabetic (Non-D) spontaneously hypertensive rats (SHR) and diabetic SHR treated with vehicle (D) or valsartan (Val, 5 mg/kg, ip) or angiotensin(1-7) [ANG(1-7), 500 μg/kg ip] for 4 wk. A: body weight. B: systolic blood pressure (SBP). C: diastolic blood pressure (DBP). Values were expressed as means ± SE, n = 8. ###P < 0.001 vs. Non-D group. *P < 0.05, **P < 0.01 vs. Dia group.
| Week 0, mmol/L | Week 2, mmol/L | Week 4, mmol/L | |
|---|---|---|---|
| Non-D | 5.3 ± 0.4 | 6.8 ± 0.7 | 6.1 ± 0.5 |
| Dia | 29.3 ± 2.1 | 31.5 ± 0.9 | >33 |
| Val | 27.4 ± 2.4 | >33 | >33 |
| ANG(1-7) | 28.5 ± 2.0 | 27.5 ± 2.4 | >33 |
Effects of Valsartan and ANG(1-7) on Bone Metabolism Markers of SHR
To better understand the potential in vivo effects of angiotensin receptor blocker (ARB) and ANG(1-7) on bone tissue, contents of minerals and bone turnover markers in serum were measured. Significant reduction in calcium (Fig. 9A, P < 0.05), phosphorus (P < 0.05), and PINP (Fig. 9B, P < 0.001) and elevation in TRAcP-5b (Fig. 9C, P < 0.05) were found in the serum of the diabetic SHR. ANG(1-7) (P < 0.05) and valsartan (P < 0.05), respectively, enhanced the circulating levels of calcium and phosphorus, and both of them decreased the serum level of TRACP-5b (P < 0.05) as compared with those in the diabetic SHR group.
Figure 9.Serum chemistries of non-diabetic (Non-D) spontaneously hypertensive rats (SHR) and diabetic SHR treated with vehicle (D) or valsartan (Val, 5 mg/kg ip) or angiotensin(1-7) [Ang(1-7), 500 μg/kg ip] for 4 wk. A: serum level of calcium (Ca) and phosphorus (P). B: procollagen type I N-propeptide (PINP). C: tartrate-resistant acid phosphatase 5b (TRAcP-5b). Values were expressed as means ± SE, n = 8. #P < 0.05, ###P < 0.001 vs. Non-D group. *P < 0.05 vs. Dia group.
HE Staining and TRAP Staining
HE staining (Fig. 10A) was conducted to evaluate the effects of drug treatment on the epiphyseal region of distal femoral metaphysis. The loss of trabecular bone quantity shown by the decrease in BA/TA (P < 0.01) was clearly observed at the secondary spongiosa zone of the distal femur in the diabetic SHR group as compared with the nondiabetic control. Treatment with ANG(1-7), but not valsartan, resulted in a rise of BA/TA (P < 0.01) at the distal end of the femur. TRAP staining (Fig. 10B), indicating the existence of matured osteoclasts at the distal femoral end, demonstrated that both ANG II (P < 0.05) and ANG(1-7) (P < 0.01) could remarkably reverse the hyperglycemia-induced enhancement on the osteoclast surface.
Figure 10.Histological images of the distal femoral metaphysis measured by hematoxylin and eosin (HE) staining and tartrate-resistant acid phosphatase (TRAP) staining in the non-diabetic (Non-D) group and diabetic groups treated with vehicle (D) or valsartan (Val) or angiotensin(1-7) [Ang(1-7)] for 4 wk. A: the representative HE image (top, magnification ×20) and the quantitative data (bottom) expressed as trabecular bone area over total area (BA/TA). B: the representative TRAP image (top, magnification ×100) and the quantitative data (bottom) expressed as osteoclasts-covered bone surface (OcS/BS). Values were expressed as means ± SE, n = 8. ##P < 0.01 vs. Non-D group.*P < 0.05, **P < 0.01 vs. Dia group.
Bending Test and Micro-CT Analysis
Mechanical strength at the tibial diaphysis and trabecular BMD at the proximal tibial metaphysis were, respectively, assessed by a three-point bending test and micro-CT. Ultimate force (Fig. 11A) and trabecular BMD (Fig. 11B) were much lower in the diabetic group (P < 0.05) compared with those in the nondiabetic group. Administration to diabetic SHR with ANG(1-7), but not valsartan, markedly elevated the ultimate force (P < 0.05) and BMD (P < 0.05), suggesting the beneficial effects of ANG(1-7) on cortical biomechanical strength and trabecular bone mass.
Figure 11.Mechanical strength at the diaphysis of the tibia and trabecular bone mass at the proximal metaphysis of tibia measured by three-point bending test and microcomputed tomography, respectively, in the non-diabetic (Non-D) group and diabetic groups treated with vehicle (D) or valsartan (Val) or angiotensin(1-7) [Ang(1-7)] for 4 wk. A: ultimate force. B: bone mineral density (BMD). Values were expressed as means ± SE, n = 8. #P < 0.05 vs. Non-D group.*P < 0.05 vs. Dia group.
Protein Expression of Renin and ANG II in Bone Tissue
Expression of renin and content of ANG II were measured in the tibia to investigate the effects of valsartan and ANG(1-7) on key components within RAS in bone tissue (Fig. 12A). Protein expression of renin (Fig. 12B, P < 0.05) and content of ANG II (P < 0.01) were much higher in the vehicle-treated diabetic group compared with those in the nondiabetic group. Valsartan treatment further upregulated (P < 0.05) the expression of renin and the content of ANG II in the tibia of diabetic SHR. On the contrary, ANG(1-7) normalized (P < 0.01) renin expression and ANG II content to the level of the nondiabetic group.
Figure 12.Protein expression of renin and angiotensin II in the tibia of the non-diabetic (Non-D) group and diabetic groups treated with vehicle (D) or valsartan (Val) or angiotensin(1-7) [Ang(1-7)] for 4 wk. A: immunoblotting bands. B: the densitometric quantification of the protein bands. Values were expressed as means ± SE, n = 8. #P < 0.05, ##P < 0.01 vs. Non-D group. *P < 0.05, **P < 0.01 vs. Dia group.
DISCUSSION
Long-term hyperglycemia produces detrimental effects on multiple tissues, thereafter diabetic complications are developed. It was estimated that ∼70% of the total direct medical costs for DM were attributed to diabetes-related complications (18). Significant evidence from the past decades revealed that RAS functioned locally in tissues beyond blood pressure control (19, 20). Remarkable experimental and clinical data implicated that ANG II, an active mediator within RAS, induced tissue damages associated with diabetes (19, 21). Thus, the relevance of RAS inhibitors that might control homeostatic conditions in tissues has received increasing attention lately. In addition, the cascade of ANG(1-7), a newly described RAS active peptide, is recognized as a protective arm counterbalancing ANG II action (15). In the present study, we investigated the differential responses of osteoblasts and ostoclasts to ANG II and ANG(1-7) with exposure to high glucose level and studied their actions on the bone of male spontaneously hypertensive rats with diabetes.
This study found that the blocking of Angiotensin II type 1 receptor by OLM markedly abolished the suppressive effects of ANG II on ALP activity and calcium nodule formation in MC3T3-E1 cells with exposure to high glucose level, suggesting the vital role of AT1R in mediating the action of ANG II in osteoblastogenesis, in accordance with the decrease in ALP activity, the number and the total area of mineralized nodules in rat calvarial osteoblastic cells by ANG II (22, 23). Of note, this study showed no marked influence of ANG II on osteoblastic functional markers under low glucose level, demonstrating there were more potential risks for bone formation impairment associated with diabetes, especially with the enhancement in local RAS activity. In addition, ANG II inhibited the expression of osteoblast-specific proteins such as RUNX2, COL-1, and OCN, regardless of glucose level in culture. This was in agreement with the decreased mRNA expression of OCN and serum level of OCN in ANG II-treated osteoblastic cells (22) and in patients with type 1 diabetes (24), respectively. Combined with a previous report that concluded ANG II stimulated expressions of matrix metalloproteinase (MMP)-3 and -13 in osteoblastic ROS17/2.8 cells (25), ANG II is postulated to be able to regulate osteoblastogenesis and degradation of bone extracellular matrix.
On the other hand, ANG(1-7) stimulated osteoblastogenesis as shown by the rise in ALP activity and calcium deposit formation and the upregulation in protein expression of RUNX2, COL-1, and OCN, via G-protein-coupled receptor Mas since the selective Mas receptor antagonist A779 could oppose these effects of ANG(1-7) on MC3T3-E1 cells. A recent study also found that treatment of primary osteoblasts from the calvaria of neonatal Wistar rats with ANG(1-7) boosted ALP activity and matrix synthesis and upregulated transcription of osterix, OCN, and COL-1 in a Mas receptor-dependent manner (15). Intriguingly, another study disclosed that ANG(1-7) attenuated the osteogenic transition of vascular smooth muscle cells by decreasing the expression of osteogenesis proteins (RUNX2 and OCN) in aortas with vascular calcification (26). Thus, the effect of ANG(1-7)/Mas axis on osteogenesis markers might be dependent on the type of tissue, and the present in vitro study showed that this axis might be a beneficial RAS cascade in bone formation besides in cardiovascular system (3, 5).
The present osteoblast studies showed that the osteoblastic effect of ANG II and ANG(1-7) was mostly seen in high glucose condition, which could inhibit the proliferation and differentiation of osteoblasts through pyroptosis pathway (27). In view of this cellular mechanism, distinguished from low glucose microenvironment, whether ANG II and ANG(1-7) could interfere with pyroptosis of osteoblasts under high glucose condition remains to be clarified.
The response of osteoclasts to ANG II and ANG(1-7) was further determined under both low glucose and high glucose levels in medium. Technically, tartrate-resistant acid phosphatase staining illustrates the existence of matured osteoclasts and pit formation assay demonstrates the osteoclast-involved resorption activity (28). Regardless of glucose level, treatment with ANG II and ANG(1-7) could dramatically induce and reduce, respectively, the matured osteoclast number and the bone resorption area, and AT1R blocker and Mas receptor antagonist could completely reverse these effects. To further explain the effects of ANG II and ANG(1-7) on bone resorption, the expression of osteoclast-specific proteins was measured. Cathepsin K and MMP9 are synthesized and secreted from osteoclast and are responsible for resorbing organic matrix in bone tissue. Similarly, our study elucidated that MMP9 protein expression was distinctly promoted and inhibited by ANG II and ANG(1-7), respectively. Under high glucose level, ANG II and ANG(1-7) could upregulate and downregulate cathepsin K protein expression, respectively. These results supported that ANG II/AT1R cascade and ANG(1-7)/Mas axis might, respectively, destroy and protect the skeletal system.
Osteoclast differentiation is coordinated by the binding of M-CSF and RANKL to their respective receptors c-Fms and RANK on the surface of osteoclast precursors (29). It was well documented that MAPK and Akt signaling pathways play essential roles in M-CSF- and RANKL-induced osteoclast activation and differentiation (30, 31). The current study showed a differential pattern in MAPK activation induced by ANG II, which could further promote an immediate phosphorylation of p38 at 15 min and JNK at 30 min under high glucose level. Consistently, the expressions of ANG II and phospho-p38 were all significantly increased at the lumbar dorsal spinal cord of diabetic mice (32). Our study also clarified that different signaling pathways (p38 and JNK) contributed to the sustained MAPK activation induced by ANG II in osteoclasts incubated with high glucose. In addition, this study found that ANG(1-7) persistently exerted inhibitory effects on phosphorylation of p38 and ERK when osteoclasts were treated with high glucose. The p38 signaling pathway participates likewise in bone metabolism regulated by circadian rhythm (33) and the blockade of p38 signaling pathway could block osteoclast differentiation (34, 35). Thus, the p38 pathway might be a potential therapeutic target for osteoporotic bone defects due to increased osteoclast formation and activation associated with hyperglycemia.
Osteoporosis, diabetes, and hypertension are common age-related disorders which together account for significant morbidity and mortality in older adults (36, 37). Given the inhibitory effects of ANG II on osteoblastic function and the stimulatory effects of ANG II on osteoclastic activity as well as the beneficial effects of ANG(1-7) on bone observed in in vitro studies with the introduction of high glucose in culture medium, we further investigated if AT1R blocker and ANG(1-7) could potentially exert osteoprotective effects on SHR with hyperglycemia. As expected, 4-wk treatment with valsartan effectively ameliorated SBP and DBP of SHR. However, intraperitoneal injection of ANG(1-7), contrary to the results that showed attenuation of mean arterial pressure (MAP) and inhibition of ANG II-induced elevation in MAP (38) by the intravenous injection of ANG(1-7), had no effect on the blood pressure of SHR. Such contradictory findings might be attributed to the differences in drug administration system that should be explored in further study. Taken together, the present study demonstrated that ANG(1-7) did not alter the blood pressure or fasting blood glucose of diabetic SHR, therefore the therapeutic effects resulting from the drug treatment were unlikely to be mediated by reduced hyperglycemia or hypertension.
ANG(1-7) treatment corrected the decrease in calcium content in circulation and the elevation in TRAcP-5b level in serum as well as the increase in osteoclasts surface at trabecular bone, which might be attributed to its repressive effects on osteoclast maturation and resorptive activity as shown in this in vitro study. Importantly, ANG(1-7) dramatically recovered trabecular bone mass (BMD) and increased mechanical strength (ultimate force) of the cortical bone in diabetic SHR. Consistently, ANG(1-7) ameliorated the structural and biochemical alterations of rats with ovariectomy-induced osteoporosis (16). So far, only few studies have been reported on the health beneficial effects of ANG(1-7) on the skeletal system. We provided the first in vivo evidence about the bone-sparing effect (trabecular bone and cortical bone) of ANG(1-7) in rats with hypertension and hyperglycemia.
In line with the action of ANG II on osteoclasts in this study, AT1R blocker valsartan could increase serum phosphorus level and alleviate serum TRAcP-5b level and TRAP-positive staining in bone microenvironment. However, neither valsartan nor ANG(1-7) could influence the circulating level of PINP, one of the markers of bone metabolism, thus the exact mechanism for the in vivo effects of ANG(1-7) and valsartan on mineral metabolites (calcium and phosphorus) and bone metabolism need to be further explored. Unexpectedly, we did not find any beneficial effects of valsartan on the biological properties of trabecular bone and cortical bone in diabetic SHR. In accordance with this study, one clinical study with a large sample size of community-dwelling older adults demonstrated that the use of ARBs did not have any significant overall effects on bone loss in older men (39).
It has been argued that ARBs could not completely block the RAS cascade due to the disruption of the feedback inhibition of renin production (40). The increase in renin activity stimulates the conversion of ANG I and ultimately ANG II, consequently this feedback largely limits the efficacy of RAS inhibitors (41). The increased renin can also act through the prorenin/renin receptor, which may cause tissue damages independent of ANG II (42). Consistently, this study using SHR with diabetes showed that valsartan led to a remarkable rise in protein expression of renin and ANG II in bone, which might, at least partially, account for the unbenefited effect of valsartan on bone phenotype. Apart from the angiotensin receptor blocker activity, some unique ARBs such as telmisartan (43, 44) and candesartan (45) are also partial agonists of peroxisome proliferator-activated receptor gamma (PPAR-γ), which is a nuclear hormone receptor whose activation results in bone loss (46). Whether valsartan could act on bone via regulating PPAR-γ pathway remains unknown.
Surely, the suppressive effects of ANG(1-7) on renin expression and ANG II content might contribute to its osteoprotective effects in diabetic SHR. Similar observations of ANG(1-7) reducing the abundance of ANG II in femoral bone head of ovariectomy-induced osteoporotic rats (16) and in aorta of rats with vascular calcification (26) have been reported, fully suggesting that ANG(1-7)-mediated signaling is a protective arm of RAS to prevent tissue damage.
In the present in vitro study, the potential effect of osmolarity on tested parameters was dismissed despite high glucose in culturing medium in this study. In addition, prewashing with PBS to remove bone marrow before protein extraction from rat bones did not rule out the possibility of renin and/or angiotensin being taken up from the circulation and retained in the bone. These two limitations should be considered and clarified in future studies.
In summary, the present study demonstrated the inhibition and the promotion of ANG II on osteoblastic function and osteoclastogenesis, respectively, under exposure of high glucose, but ANG II type 1 receptor blocker did not display any osteoprotective effects due to, at least partially, the feedback enhancement in the expression of renin and content of ANG II in diabetic SHR. In vitro and in vivo studies provided evidence for the first time that ANG(1-7) was able to stimulate osteogenesis and inhibit osteoclastic resorption, consequently exerting protection against osteoporosis in SHR with hyperglycemia. How the cross talk and interaction between the dual axis systems [ANG II/AT1R and ANG(1-7)/Mas receptor] of RAS contribute to the management of bone metabolism needs to be further studied for a better understanding of the molecular mechanism involved in bone metabolic diseases and for exploring novel drug candidates for treatment of osteoporosis.
GRANTS
This work was supported in part by National Natural Science Foundation of China (81774329, 82074468), Essential Drug Research and Development (2019ZX09201004-003-032), National Key R&D Program (2018YFC1704302) and Program for Innovative Research Team (2015RA4002) from Ministry of Science and Technology of China, Hundred Talents Program from Shanghai Municipal Commission of Health and Family Planning (2018BR03), Program of Shanghai Academic Research Leader (19XD1423800), Natural Science Foundation of Shanghai (17ZR1430800), and Sanming Project of Medicine in Shenzhen (SZSM201808072).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Y.-J.W. and Y.Z. conceived and designed research; N.-N.S., J.-L.Z., W.-X.L., Y.L., Y.-F.W., W.S., F.-H.L., and W.-P.L. performed experiments; N.-N.S., J.-L.Z., and C.-C.P. analyzed data; J.-L.Z., C.-C.P., and F.-H.L. interpreted results of experiments; N.-N.S. prepared figures; Y.Z. drafted manuscript; Y.Z. edited and revised manuscript; Y.-J.W. approved final version of manuscript.
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