SOST mRNA Research Articles

Toll-like receptor-2 induced inflammation causes local bone formation and activates canonical Wnt signaling.

It is well established that inflammatory processes in the vicinity of bone often induce osteoclast formation and bone resorption. Effects of inflammatory processes on bone formation are less studied. Therefore, we investigated the effect of locally induced inflammation on bone formation. Toll-like receptor (TLR) 2 agonists LPS from Porphyromonas gingivalis and PAM2 were injected once subcutaneously above mouse calvarial bones. After five days, both agonists induced bone formation mainly at endocranial surfaces. The injection resulted in progressively increased calvarial thickness during 21 days. Excessive new bone formation was mainly observed separated from bone resorption cavities. Anti-RANKL did not affect the increase of bone formation. Inflammation caused increased bone formation rate due to increased mineralizing surfaces as assessed by dynamic histomorphometry. In areas close to new bone formation, an abundance of proliferating cells was observed as well as cells robustly stained for Runx2 and alkaline phosphatase. PAM2 increased the mRNA expression of Lrp5, Lrp6 and Wnt7b, and decreased the expression of Sost and Dkk1. In situ hybridization demonstrated decreased Sost mRNA expression in osteocytes present in old bone. An abundance of cells expressed Wnt7b in Runx2-positive osteoblasts and ß-catenin in areas with new bone formation. These data demonstrate that inflammation, not only induces osteoclastogenesis, but also locally activates canonical WNT signaling and stimulates new bone formation independent on bone resorption.

Advanced glycation end products impair bone marrow mesenchymal stem cells osteogenesis in periodontitis with diabetes via FTO-mediated N6-methyladenosine modification of sclerostin

BackgroundDiabetes mellitus (DM) and periodontitis are two prevalent diseases with mutual influence. Accumulation of advanced glycation end products (AGEs) in hyperglycemia may impair cell function and worsen periodontal conditions. N6-methyladenosine (m6A) is an important post-transcriptional modification in RNAs that regulates cell fate determinant and progression of diseases. However, whether m6A methylation participates in the process of periodontitis with diabetes is unclear. Thus, we aimed to investigate the effects of AGEs on bone marrow mesenchymal stem cells (BMSCs), elucidate the m6A modification mechanism in diabetes-associated periodontitis.MethodsPeriodontitis with diabetes were established by high-fat diet/streptozotocin injection and silk ligation. M6A modifications in alveolar bone were demonstrated by RNA immunoprecipitation sequence. BMSCs treated with AGEs, fat mass and obesity associated (FTO) protein knockdown and sclerostin (SOST) interference were evaluated by quantitative polymerase chain reaction, western blot, immunofluorescence, alkaline phosphatase and Alizarin red S staining.ResultsDiabetes damaged alveolar bone regeneration was validated in vivo. In vitro experiments showed AGEs inhibited BMSCs osteogenesis and influenced the FTO expression and m6A level in total RNA. FTO knockdown increased the m6A levels and reversed the AGE-induced inhibition of BMSCs differentiation. Mechanically, FTO regulated m6A modification on SOST transcripts, and AGEs affected the binding of FTO to SOST transcripts. FTO knockdown accelerated the degradation of SOST mRNA in presence of AGEs. Interference with SOST expression in AGE-treated BMSCs partially rescued the osteogenesis by activating Wnt Signaling.ConclusionsAGEs impaired BMSCs osteogenesis by regulating SOST in an m6A-dependent manner, presenting a promising method for bone regeneration treatment of periodontitis with diabetes.

SIRT6-PAI-1 axis is a promising therapeutic target in aging-related bone metabolic disruption

The mechanistic regulation of bone mass in aged animals is poorly understood. In this study, we examined the role of SIRT6, a longevity-associated factor, in osteocytes, using mice lacking Sirt6 in Dmp-1-expressing cells (cKO mice) and the MLO-Y4 osteocyte-like cell line. cKO mice exhibited increased osteocytic expression of Sost, Fgf23 and senescence inducing gene Pai-1 and the senescence markers p16 and Il-6, decreased serum phosphate levels, and low-turnover osteopenia. The cKO phenotype was reversed in mice that were a cross of PAI-1-null mice with cKO mice. Furthermore, senescence induction in MLO-Y4 cells increased the Fgf23 and Sost mRNA expression. Sirt6 knockout and senescence induction increased HIF-1α binding to the Fgf23 enhancer sequence. Bone mass and serum phosphate levels were higher in PAI-1-null aged mice than in wild-type mice. Therefore, SIRT6 agonists or PAI-1 inhibitors may be promising therapeutic options for aging-related bone metabolism disruptions.

All-trans Retinoic Acid and Beta-Carotene Increase Sclerostin Production in C2C12 Myotubes.

Sclerostin is a protein secreted by osteocytes whose encoding gene SOST is regulated by mechanical stimuli, cytokines, and all-trans retinoic acid (ATRA) and mediates antianabolic effects on bone formation as an inhibitor of the canonical Wnt/β-catenin pathway. Interestingly, skeletal muscle has recently been identified as another source of sclerostin, suggesting that the musculature may play an important role in maintaining bone mass. However, regulators of muscular SOST expression are virtually unknown. This study investigates the influence of ATRA and the provitamin A derivative beta-carotene (β-C) on sclerostin synthesis in muscle cells. The impact of ATRA, its synthetic analog TTNPB, and β-C on Sost transcription was analyzed by qRT-PCR in C2C12 myotubes and the secreted sclerostin protein by ELISA. ATRA strongly increases the sclerostin synthesis in C2C12 myotubes in a dose-dependent manner. The stimulating effect of ATRA and TTNPB on Sost is largely reduced in the presence of the retinoic acid receptor inhibitor AGN193109. β-C also increases the Sost expression, but this effect vanishes when β-C is coincubated with beta-carotene 15,15'-monooxygenase 1 (BCMO1)-specific siRNA. Thus, ATRA is a potent stimulator of sclerostin release in muscle cells. β-C can also increase Sost mRNA abundance, but this effect depends on the conversion to a retinoid.

FTY720 administration results in a M2 associated immunoregulatory effect that positively influences the outcome of alveolar bone repair outcome in mice.

The alveolar bone repair process may be influenced by multiple local and systemic factors, which include immune system cells and mediators. Macrophages allegedly play important roles in the repair process, and the transition of an initial inflammatory M1 profile into a pro-reparative M2 profile theoretically contributes to a favorable repair outcome. In this context, considering immunoregulatory molecules as potential targets for improving bone repair, this study evaluated the role of the immunoregulatory molecule FTY720, previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups submitted to tooth extraction and maintained under control conditions or treated with FTY720 were evaluated by microtomographic (μCT), histomorphometric, immunohistochemical and molecular analysis to characterize healing and host response features at 0, 1, 3, 7 and 14days. Our results demonstrated that the FTY720 group presented higher bone tissue density, higher bone tissue volume, greater tissue volume fraction, greater number and thickness of trabeculae and a higher number of osteoblasts and osteoclasts than the control group. Accordingly, the bone markers BMP2, BMP7, ALPL, SOST and RANK mRNA expressions increased in the FTY720 treated group. Furthermore, the levels of FIZZ, ARG2 and IL-10 mRNA increased in the FTY720 group together with the presence of CD206+ cells, suggesting that the boost of bone formation mediated by FTY720 involves an increased polarization and activity of M2 macrophages in healing sites. Thus, our results demonstrate that FTY720 favored the process of alveolar bone repair, probably trough a strengthened M2 response, associated with an increased expression of markers osteogenic differentiation and activity markers. Immunoregulatory strategies based in the modulation of macrophage polarization profile can comprise effective tools to improve the bone repair process.

An Insight Into the Association of Sclerostin With Insulin Sensitivity and Glycemic Parameters in Male Indian Prediabetic and Diabetic Population.

BackgroundType 2 diabetes (T2D) is increasing day by day and creating a huge financial and social burden on the Indian population. Insulin resistance results in hyperglycemia, a condition that eventually causes prediabetes and Type 2 diabetes. The etiopathogenesis of T2D is still not clearly defined. Wnt signaling pathway is involved in pancreas development, islet function, insulin production, and secretion. Recent studies show that sclerostin, a Wnt signaling inhibitor, is associated with diabetes. The sclerostin level is altered as a function of race and ethnicity. However, no study has been conducted to observe the sclerostin level in prediabetic and diabetic individuals in the Indian population.ObjectivesThe main objectives of the study are: to determine whether sclerostin is associated with glycemic parameters, serum insulin levels, insulin resistance/ sensitivity, beta-cell function, and adipose tissue insulin resistance (Adipo-IR).MethodsThis observational study was carried out at a tertiary care hospital, in Rishikesh, Uttarakhand, India. Individuals with T2D and prediabetes and healthy references were included in this study. Sclerostin and free fatty acids (FFA) were measured with the enzyme-linked immunosorbent assay (ELISA), and blood sugar, insulin, and glycated haemoglobin (HbA1c) were measured by the hexokinase, chemiluminescent, and chromatography methods, respectively. Messenger RNA (mRNA) was quantified by real-time polymerase chain reaction (PCR) using the SYBR Green protocol. Adipo-IR, homeostasis model assessment-estimated insulin resistance (HOMA-IR), homeostasis model assessment of β-cell function (HOMA-B), quantitative insulin sensitivity check index (QUICKI), and single point insulin sensitivity estimator (SPISE) indices were calculated.ResultsA total of 171 study participants were enrolled in type 2 diabetes, prediabetes, and controls groups, having 57 each in the group. There was a gradual increase in sclerostin levels from healthy [242.12(158.44)] to prediabetes [256.06(299.65)] and diabetes [465.76 (735.71)] with a significant (<0.001) difference from healthy reference. Sclerostin showed a significant positive correlation with fasting blood sugar (r=0.200; p=0.009), HbA1c (r=0.394; p<0.001) and free fatty acids (r=0.205; p=0.007) in total study participants. The SPISE index showed a significant positive correlation (r=0.269, p=0.043) in the prediabetic group. SOST, GLUT4, and insulin receptor (IR) mRNA expression all corroborate with the glycemic status.ConclusionSignificantly higher expression of sclerostin (both protein and gene) in newly diagnosed T2D and prediabetes male patients, as well as significant association with SPISE index, suggest that sclerostin might be an indicator of pathophysiology related to insulin resistance, which is a characteristic feature of diabetes mellitus. However, the identification of causal relationships would warrant a large-scale prospective cohort study.

Dynamic alternations of RANKL/OPG ratio expressed by cementocytes in response to orthodontic‑induced external apical root resorption in a rat model.

The aim of the present study was to investigate the alterations in the formation of cementocytes in response to orthodontic forces and to evaluate the contribution of these cells in the biological changes of tooth movement and associated root resorption. A total of 90 Sprague Dawley rats were randomly assigned to the control, high force, and low force groups. Intrusion forces of 10 and 50 g were applied on the rat molar to induce tooth intrusion. The tooth movement was observed from 0 to 14 days by micro-computed tomography, bone histometric analysis, tartrate-resistant acid phosphatase staining, as well as reverse transcription-quantitative PCR and immunofluorescence staining assays. The results suggested that under low force conditions, osteoclasts were distributed at a higher frequency on the bone side than on the root side. Under high force conditions, both sides suffered osteoclast infiltration. In the low force group, the cementocytes exhibited downregulated sclerostin (SOST) and osteoprotegerin (OPG) mRNA levels and a lower receptor activator of nuclear factor-κB ligand (RANKL)/OPG ratio over a certain period of time. The expression levels of these genes were lower compared with those of the osteocytes at each time-point. In the high force group, both cementocytes and osteocytes upregulated the SOST and RANKL/OPG ratio on days 7 and 14, while the cementocytes expressed higher levels of SOST mRNA than those noted in the osteocytes. These data suggested that cementocytes responded to the orthodontic force via modulation of the RANKL/OPG ratio and SOST expression. The biological response of cementocytes contributed to the mechanotransduction and homoeostasis of the roots under compression. Excessive forces may act as a negative factor of this regulatory role. These results expand our knowledge on the function of cementocytes.

Non-canonical Wnt signaling participates in Jagged1-induced osteo/odontogenic differentiation in human dental pulp stem cells

Osteoblast differentiation requires the interaction of various cell signaling pathways to modulate cell responses. Notch and Wnt signaling are among the crucial pathways that control numerous biological processes, including osteo/odontogenic differentiation. The aim of the present study was to examine the involvement of Wnt signaling in the Jagged1-induced osteo/odontogenic differentiation in human dental pulp stem cells (hDPSCs). The Wnt-related gene expression was analyzed from publicly available data of Jagged1-treated human dental pulp cells. The mRNA expression of Wnt ligands (WNT2B, WNT5A, WNT5B, and WNT16) and Wnt inhibitors (DKK1, DKK2, and SOST) were confirmed using real-time polymerase chain reaction. Among the Wnt ligands, WNT2B and WNT5A mRNA levels were upregulated after Jagged1 treatment. In contrast, the Wnt inhibitors DKK1, DKK2, and SOST mRNA levels were downregulated. Recombinant WNT5A, but not WNT2B, significantly promoted in vitro mineral deposition by hDPSCs. Wnt signaling inhibition using IWP-2, but not DKK1, inhibited Jagged1-induced alkaline phosphatase (ALP) activity, mineralization, and osteo/odontogenic marker gene expression in hDPSCs. In conclusion, Jagged1 promoted hDPSC osteo/odontogenic differentiation by modulating the non-canonical Wnt pathway.

Novel roles of tendon in the postnatal growth and expansion of TMJ condyle

The Temporomandibular joint (TMJ) is one of the most complex joints in the human body. TMJ is composed of the temporal bone, a disc and a movable mandibular condyle with abundant tendon attachments. Tendon has been thought to play the sole function of transmitting muscle forces to stabilize joints, yet it is largely unclear why tendon undergoes ectopic ossification in trauma or diseases, and whether it has any direct contribution to skeletal formation. This study aimed to investigate the full biological significance of tendon in TMJ growth. We first discovered that the TMJ condyle is composed of a well‐established cartilage head and an overlooked “bony head” that grows after birth and continuously expands throughout the lifespan with little signs of remodeling. Mouse X‐ray images (Fig.1a) showed little change in the cartilage head’s volume but a continuous expansion in the bony head’s mass with a low mineral content from 1 to 5 months (Fig.1b). Toluidine blue staining showed TMJ condyle had a large area of tendon attachment extending down to ramus (Fig.1c, white dotted line in lower magnification), defined by regions of tendon, interface, and TFB (Fig.1c1). The TFB morphology was distinct from endosteum‐formed bone (EFB, Fig.1c1), cartilage‐formed bone (CFB, Fig.1c2, rich in cartilage residual), or periosteum‐formed bone (PFB, Fig.1c3) in cell shape and distribution, and ECM. TEM images further revealed that the osteocytes in the TFB were large in size, irregular in shape, had small nuclei but numerous ERs and Golgi complexes, and were embedded in ECM rich in fibropositors. In contrast, the osteocytes in EFB, CFB or PFB were spindle‐shaped with larger nuclei but fewer ERs and Golgi complexes (Fig.1d). To reveal the cell source of the bony head, cell lineage tracing were used. Tracing data showed that most CFB cells originate from Col10a1+ hypertrophic chondrocytes, whereas the interface and TFB were derived from Scx+ cells (Fig.1e). RNAscope displayed high levels of Thbs4 (Thrombospondin‐4, a tendon marker) and SOST (a potent inhibitor of Wnt signaling secreted by osteocytes) mRNA in TFB at bony head (Fig.1f). The Scx‐CreERT2 tracing combined with IHC staining showed TFB maintained a mixed ECM of bone (Col1), cartilage (Aggrecan) and tendon (Periostin, Fig.1g). To further determine the role of tendon lineage in condyle expansion, we generated Scx‐CreERT2; R26RDTA (carrying a loxP‐flanked stop cassette associated with an attenuated diphtheria toxin fragment A, DTA, for the ablation of cells when Cre is active). Deletion of Scx+ cells greatly reduced the size of bony head (Fig.1h) and the thickness of interface with few Scx+/Col1+ bone cells in P28 DTA mice (Fig.1i); In conclusion, our study tendon cells, beyond their conventional role in joint movement, are key players for the postnatal growth and expansion of TMJ condyle (Fig.1j).

Generation of two multipotent mesenchymal progenitor cell lines capable of osteogenic, mature osteocyte, adipogenic, and chondrogenic differentiation

Mesenchymal progenitors differentiate into several tissues including bone, cartilage, and adipose. Targeting these cells in vivo is challenging, making mesenchymal progenitor cell lines valuable tools to study tissue development. Mesenchymal stem cells (MSCs) can be isolated from humans and animals; however, obtaining homogenous, responsive cells in a reproducible fashion is challenging. As such, we developed two mesenchymal progenitor cell (MPC) lines, MPC1 and MPC2, generated from bone marrow of male C57BL/6 mice. These cells were immortalized using the temperature sensitive large T-antigen, allowing for thermal control of proliferation and differentiation. Both MPC1 and MPC2 cells are capable of osteogenic, adipogenic, and chondrogenic differentiation. Under osteogenic conditions, both lines formed mineralized nodules, and stained for alizarin red and alkaline phosphatase, while expressing osteogenic genes including Sost, Fgf23, and Dmp1. Sost and Dmp1 mRNA levels were drastically reduced with addition of parathyroid hormone, thus recapitulating in vivo responses. MPC cells secreted intact (iFGF23) and C-terminal (cFGF23) forms of the endocrine hormone FGF23, which was upregulated by 1,25 dihydroxy vitamin D (1,25D). Both lines also rapidly entered the adipogenic lineage, expressing adipose markers after 4 days in adipogenic media. MPC cells were also capable of chondrogenic differentiation, displaying increased expression of cartilaginous genes including aggrecan, Sox9, and Comp. With the ability to differentiate into multiple mesenchymal lineages and mimic in vivo responses of key regulatory genes/proteins, MPC cells are a valuable model to study factors that regulate mesenchymal lineage allocation as well as the mechanisms that dictate transcription, protein modification, and secretion of these factors.

Free-fall landing and interval running have different effects on trabecular bone mass and microarchitecture, serum osteocalcin, biomechanical properties, SOST expression and on osteocyte-related characteristics.

The effects of treadmill interval training (IT) and free-fall exercise were evaluated on bone parameters including osteocyte related characteristics. Thirty-eight 4-month-old male Wistar rats were randomly divided into a control (C) group and exercise groups: IT, 10 free-fall impacts/day with a 10-s (FF10) or 20-s interval between drops (FF20), 5 days/week, for 9 weeks. We assessed bone mineral density (BMD); microarchitecture by µCT; mechanical strength by a 3-point bending test; density and occupancy of the osteocyte lacunae by toluidine blue staining; osteocalcin and NTx systemic levels by ELISA; and bone tissue Sost messenger RNA (mRNA) expression by RT-PCR. NTx levels were significantly lower in exercise groups as compared with the C group. In exercise groups the Sost mRNA expression was significantly lower than in C. Tb.N was significantly higher for IT and FF20 compared with the C group. Tb.Sp was significantly lower in FF10 compared with the C group. Both IT and FF20 were associated with higher tibial lacunar density as compared with FF10. compared with FF10, IT fat mass was lower, while tibial osteocyte lacunae occupancy and systemic osteocalcin level were higher. All exercise modes were efficient in reducing bone resorption. Both IT and free-fall impact with appropriate recovery periods, which may be beneficial for bone health and osteocyte-related characteristics. Novelty: Interval training is beneficial for bone mineral density. Exercises decreased both bone resorption and inhibition of bone formation (Sost mRNA). Longer interval recovery time favors osteocyte lacunae density.

Skeletal Effects of Inducible ERα Deletion in Osteocytes in Adult Mice.

Estrogen is known to regulate bone metabolism in both women and men, but substantial gaps remain in our knowledge of estrogen and estrogen receptor alpha (ERα) regulation of adult bone metabolism. Studies using global ERα-knockout mice were confounded by high circulating sex-steroid levels, and osteocyte/osteoblast-specific ERα deletion may be confounded by ERα effects on growth versus the adult skeleton. Thus, we developed mice expressing the tamoxifen-inducible CreERT2 in osteocytes using the 8-kilobase (kb) Dmp1 promoter (Dmp1CreERT2 ). These mice were crossed with ERαfl//fl mice to create ERαΔOcy mice, permitting inducible osteocyte-specific ERα deletion in adulthood. After intermittent tamoxifen treatment of adult 4-month-old mice for 1 month, female, but not male, ERαΔOcy mice exhibited reduced spine bone volume fraction (BV/TV (-20.1%, p=0.004) accompanied by decreased trabecular bone formation rate (-18.9%, p=0.0496) and serum P1NP levels (-38.9%, p=0.014). Periosteal (+65.6%, p=0.004) and endocortical (+64.1%, p=0.003) expansion were higher in ERαΔOcy mice compared to control (Dmp1CreERT2 ) mice at the tibial diaphysis, reflecting the known effects of estrogen to inhibit periosteal apposition and promote endocortical formation. Increases in Sost (2.1-fold, p=0.001) messenger RNA (mRNA) levels were observed in trabecular bone at the spine in ERαΔOcy mice, consistent with previous reports that estrogen deficiency is associated with increased circulating sclerostin as well as bone SOST mRNA levels in humans. Further, the biological consequences of increased Sost expression were reflected in significant overall downregulation in panels of osteoblast and Wnt target genes in osteocyte-enriched bones from ERαΔOcy mice. These findings thus establish that osteocytic ERα is critical for estrogen action in female, but not male, adult bone metabolism. Moreover, the reduction in bone formation accompanied by increased Sost, decreased osteoblast, and decreased Wnt target gene expression in ERαΔOcy mice provides a direct link in vivo between ERα and Wnt signaling. © 2022 American Society for Bone and Mineral Research (ASBMR).

RANKL-Induced Increase in Cathepsin K Levels Restricts Cortical Expansion in a Periostin-Dependent Fashion: A Potential New Mechanism of Bone Fragility.

Receptor activator of nuclear factor-κΒ ligand (RANKL) is necessary and sufficient to promote osteoclastogenesis and a key pathogenic factor in osteoporosis. Failure of periosteal apposition to compensate for bone loss due to endosteal resorption further contributes to bone fragility. Whether these two processes are biologically related, however, remains unknown. Using high-resolution peripheral quantitative computed tomography (HR-pQCT), we first examined cortical bone parameters at distal radius and tibia in postmenopausal women (PMW) as well as in cadaveric human adult humeri. Increases in medullary area were negatively correlated with cortical bone volume but positively with total bone volume, and this relationship was stronger in the dominant arm, suggesting a mechanically driven process. To investigate the role of RANKL in this dual process, we used mice overexpressing huRANKL (huRANKLTg+ ). Trabecular and cortical bone volume (Ct.BV) are reduced in these mice, whereas cortical total volume (Ct.TV) is increased. In these bones, Sost mRNA levels are downregulated and periostin (Postn) mRNA levels upregulated, hence providing a positive message for periosteal bone formation. In turn, genetic deletion of Postn in huRANKLTg+ mice prevented the increase in Ct.TV and aggravated bone fragility. In contrast, cathepsin K (Ctsk) ablation improved Ct.TV in both huRANKLTg+ and wild-type (WT) mice and stimulated periosteal bone formation, while augmenting Postn protein levels. Therefore, bone strength in huRANKLTg+ /Ctsk-/- mice was restored to WT levels. These findings suggest that high levels of RANKL not only induce endosteal bone loss but may somewhat restrict periosteal bone formation by triggering periostin degradation through cathepsin K, hence providing a biological mechanism for the observed limited increase in cortical area in postmenopausal women. © 2021 American Society for Bone and Mineral Research (ASBMR).

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.

Remodeling process in bone of aged rats in response to resistance training

AimsWe investigate the effects of RT on the mechanical function, gene, and protein expression of key factors involved in bone remodeling during aging. Main methodsMale rats of 3 and 21 months of age were randomly allocated into four groups (8 per group): young sedentary (YS), young trained (YT), old sedentary (OS), and old trained (OT). RT was performed three times per week (12 weeks). Bone tenacity and stiffness were measured by biomechanical tests and mRNA levels of COL1A1, MEPE, SOST, OPG, BMP-2, PPAR-y, MMP-2-9-13, and TIMP-1 were evaluated by quantitative PCR. COL1A1 protein and MMP-2 activity were detected by western blotting and zymography assays. Key findingsAging increased stiffness, while BMP-2, OPG, COL1A1 and MMP-2 mRNA levels reduced (OS vs YS; p ≤ 0.05). RT increased the tenacity of the femur and reduced PPAR-γ regardless of age (YT vs. YS; OT vs. OS; p ≤ 0.05). RT downregulated SOST mRNA levels only in the OT group (vs. OS group, p ≤ 0.05). RT mitigated the age-associated increase in MMP-9 mRNA levels (p ≤ 0.05). In young animals, upregulation in MEPE, MMP-13, TIMP-1 were observed after RT, as well an increase in COL1A1 protein and MMP-2 activity (p ≤ 0.05). SignificanceRT improved bone tenacity independent of aging, which is relevant for mechanical function, while, at protein levels, RT upregulated MMP-2 activity and collagen 1 only in young rats. This study highlights the importance of exercise on bone health and identifies specific molecular changes in response to RT. Our findings provide insights into the mechanisms involved in age-related changes.

P0096RENAL EXPRESSION AND CIRCULATING LEVELS OF CANONICAL WNT PATHWAY INHIBITORS IN THE EXPERIMENTAL MODEL OF EARLY CHRONIC KIDNEY DISEASE

Abstract Background and Aims Molecular mechanisms implicated in the initial stages of inorganic phosphate (Pi) imbalance in chronic kidney disease (CKD) remain poorly understood. The aim of the study was to evaluate whether canonical Wnt pathway inhibitors (iWnt) involved in early response to Pi retention in CKD. Method Mild CKD was induced by 3/4 nephrectomy (NE, n=10) in spontaneously hypertensive rats (SHR) fed rat chow diet containing 0.8 % phosphate. Controls were sham operated SHR (SO, n=9). Duration of experimental exposure (NE or SO) was 4 months. Creatinine (Cr), albumin/ creatinine ratio (Alb/Cr), inorganic phosphate (Pi), fractional (FEPi) and daily Pi excretion (uPi24), intact parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho protein, sclerostin (SOST) and Dickkopf-1 (DKK1), SOST and DKK1 mRNA expression in kidney, renal interstitial fibrosis (RF) were analyzed. Results Levels of Alb/Cr, serum Cr, RF and indices of Pi exchange in the experimental model corresponded to mild CKD with Pi retention (Table). No differences were observed in serum levels of Klotho, FGF23 and PTH in experimental group vs controls (Table). Serum SOST and DKK1 concentrations were significantly higher in NE group while the kidney expression of SOST and DKK1 mRNAs decreased compared to SO (Table). Conclusion Early stages of experimental CKD are characterized by increased serum concentrations of sclerostin and Dickkopf-1 in the absence of Klotho, FGF23 or PTH levels alterations. The increase of circulating iWnt levels seems not to be associated with their renal biosynthesis and likely have a bone origin.

Effects of mild hyperbaric oxygen on osteoporosis induced by hindlimb unloading in rats.

Disuse-induced bone loss is caused by a suppression of osteoblastic bone formation and an increase in osteoclastic bone resorption. There are few data available for the effects of environmental conditions, i.e., atmospheric pressure and/or oxygen concentration, on osteoporosis. This study examined the effects of mild hyperbaric oxygen at 1317hPa with 40% oxygen on unloading-induced osteoporosis. Eighteen 8-week old male Wistar rats were randomly divided into three groups: the control for 21days without unloading and mild hyperbaric oxygen (NOR, n = 6), the unloading for 21days and recovery for 10days without mild hyperbaric oxygen (HU + NOR, n = 6), and the unloading for 21days and recovery for 10days with mild hyperbaric oxygen (HU + MHO, n = 6). The cortical thickness and trabecular bone surface area were decreased in the HU + NOR group compared to the NOR group. There were no differences between the NOR and HU + MHO groups. Osteoclast surface area and Sclerostin (Sost) mRNA expression levels were decreased in the HU + MHO group compared to the HU + NOR group. These results suggested that the loss of the cortical and trabecular bone is inhibited by mild hyperbaric oxygen, because of an inhibition of osteoclasts and enhancement of bone formation with decreased Sost expression. We conclude that exposure to mild hyperbaric oxygen partially protects from the osteoporosis induced by hindlimb unloading.

Inhibitors of canonical Wnt signaling pathway and inorganic phosphate imbalance in experimental chronic kidney disease

BACKGROUND. The molecular mechanisms of the initial stages of inorganic phosphate (Pi) metabolic disorders in chronic kidney disease (CKD) remain poorly understood.THE AIM. To test the hypothesis about changes in canonical Wnt signaling pathway inhibitors biosynthesis and a concomitant decrease in bone turnover as one of early mechanisms of Pi imbalance in CKD.MATERIAL AND METHODS. Creatinine (Cr), inorganic phosphate (Pi), serum parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), osteoprotegerin (OPG), sclerostin (SOST) and Dickkopf-1 (DKK), renal SOST and DKK mRNA expression, albuminuria (Alb), proteinuria (uTP) levels, fractional (FEPi) and daily (uPi24) Pi excretion were analyzed in SHR rats (N = 52) with 3/4 nephrectomy (NE) or sham operation (SO) and observation periods of 2, 4, and 6 months.RESULTS. Experimental model was comparable with 1-2 stages of CKD. In groups NE4 and NE6, the concentration of sPi and renal Pi excretion (FEPi and uPi24) were significantly higher vs corresponding controls SO4 (p = 0.006, p &lt;0.010) and SO6 (p = 0.002, p = 0.028). Serum concentrations of FGF23 and PTH in NE and SO animals did not change significantly. In NE4 and NE6 groups, serum SOST and DKK concentrations were significantly higher vs controls (p &lt;0.049, p &lt;0.043), while the kidney expression SOST and DKK mRNA in NE rats did not change significantly or decreased (p = 0.002, p &lt;0.011). The serum concentration of OPG was higher in the NE6 vs SO6 control (p = 0.028).CONCLUSION. The initial stages of experimental CKD are characterized by an increase in serum concentrations of Dikkopf-1, sclerostin and osteoprotegerin. The obtained data suggest the possible role of canonical Wnt signaling inhibition and reduction of bone turnover in the pathogenesis of Pi metabolic disorders in early stages of CKD.

The Influence of Pro-Inflammatory Factors on Sclerostin and Dickkopf-1 Production in Human Dental Pulp Cells Under Hypoxic Conditions.

Sclerostin (Sost) and dickkopf (Dkk)-1 are inhibitors of the Wnt signaling pathway that plays a role in regenerative processes. Hypoxia-based strategies are used for regenerative approaches, but the influence of hypoxia on Sost and Dkk-1 production in a pro-inflammatory environment is unclear. The aim of this study was to assess if pro-inflammatory molecules have an influence on Sost and Dkk-1 production in dental pulp cells (DPC) under normoxia and hypoxia. Human DPC were treated with interleukin (IL)-1β, tumor necrosis factor (TNF)α or transforming growth factor (TGF)β, with L-mimosine (L-MIM) or hypoxia or a combination. Sost and Dkk-1 mRNA and protein levels were measured with qPCR and western blot, respectively. TNFα, TGFβ, L-MIM, or combined treatment did not modulate Sost and Dkk-1. IL-1β downregulated Sost at the mRNA level. Hypoxia alone and together with inflammatory markers downregulated Dkk-1 at the mRNA level. Sost and Dkk-1 protein production was below the detection limit. In conclusion, there is a differential effect of hypoxia and IL-1β on the mRNA production of Sost and Dkk-1. Pro-inflammatory molecules do not further modulate the effects of L-MIM or hypoxia on Sost and Dkk-1 production in DPC.

Irisin Prevents Disuse-Induced Osteocyte Apoptosis.

Previous results showed that intermittently administered irisin improves bone mass in normal mice and prevents the development of disuse-induced osteoporosis and muscular atrophy in hindlimb-suspended mice, a murine model able to mimic the absence of mechanical loading. A recent study showed that irisin increases survival of osteocytes acting through integrin αV/β5 receptors. To better understand the action of irisin on these cells, we investigated the downstream signaling cascades in osteocyte-like cells (MLO-Y4) treated with recombinant irisin (rec-irisin) in vitro and we analyzed survival of osteocytes and caspase activation in cortical bone of osteoporotic mice treated with rec-irisin in vivo. Our results revealed that rec-irisin activated the MAP kinases Erk1 and Erk2 and increased the expression of the transcription factor Atf4 (2.5-fold, p < .05) through an Erk-dependent pathway in osteocytes. Some key genes expressed by MLO-Y4 cells were modulated by long-term irisin treatment, either continuously administered or given with intermittent short pulses. Interestingly, Sost mRNA was severely downregulated only upon intermittent irisin administration (10-fold, p < .001). Furthermore, rec-irisin upregulated Tfam mRNA (fourfold, p < .05) and Bcl2/Bax ratio (twofold, p < .05) in MLO-Y4 cells. By detecting caspase-9 and caspase-3, we also found that rec-irisin inhibited apoptosis induced by hydrogen peroxide and dexamethasone, respectively. In cortical bone of unloading C57BL6 mice treated with vehicle (unload-veh), irisin prevented disuse-induced reduction of viable osteocytes (+30% versus unload-veh, p < .05) and increase of empty lacunae (+110% versus unload-veh, p < .05), as well as caspase-9 (threefold, p < .05) and caspase-3 (twofold, p < .05) activations. Our findings revealed underlying mechanisms of irisin action on osteocytes, which increases their functions and exerts anti-apoptotic effects, confirming that mechanosensor cells of bone are sensitive to the exercise-mimetic myokine irisin. © 2019 American Society for Bone and Mineral Research.