Bone Mass and Osteoblast Activity Are Sex-Dependent in Mice Lacking the Estrogen Receptor α in Chondrocytes and Osteoblast Progenitor Cells

While estrogen receptor alpha (ERα) is known to be important for bone development and homeostasis, its exact function during osteoblast differentiation remains unclear. Conditional deletion of ERα during specific stages of osteoblast differentiation revealed different bone phenotypes, which were also shown to be sex-dependent. Since hypertrophic chondrocytes can transdifferentiate into osteoblasts and substantially contribute to long-bone development, we aimed to investigate the effects of ERα deletion in both osteoblast and chondrocytes on bone development and structure. Therefore, we generated mice in which the ERα gene was inactivated via a Runx2-driven cyclic recombinase (ERα^{fl/fl; Runx2Cre}). We analyzed the bones of 3-month-old ERα^{fl/fl; Runx2Cre} mice by biomechanical testing, micro-computed tomography, and cellular parameters by histology. Male ERα^{fl/fl; Runx2Cre} mice displayed a significantly increased cortical bone mass and flexural rigidity of the femurs compared to age-matched controls with no active Cre-transgene (ERα^fl/fl). By contrast, female ERα^{fl/fl; Runx2Cre} mice exhibited significant trabecular bone loss, whereas in cortical bone periosteal and endosteal diameters were reduced. Our results indicate that the ERα in osteoblast progenitors and hypertrophic chondrocytes differentially contributes to bone mass regulation in male and female mice and improves our understanding of ERα signaling in bone cells in vivo.     

Different Magnitudes of Tensile Strain Induce Human Osteoblasts Differentiation Associated with the Activation of ERK1/2 Phosphorylation

Mechanical factors are related to periprosthetic osseointegration following total hip arthroplasty. However, osteoblast response to strain in implanted femurs is unclear because of the absence of accurate stress-measuring methods. In our study, finite element analysis was performed to calculate strain distribution in implanted femurs. 0.8-3.2% tensile strain was then applied to human osteoblasts. Higher magnitudes of strain enhanced the expression of osteocalcin, type I collagen, and Cbfa1/Runx2. Lower magnitudes significantly increased ALP activity. Among these, type I collagen expression increased with the activation of ERK1/2 phosphorylation in a strain-magnitude-dependent manner. Our study marks the first investigation of osteoblast response at different magnitudes of periprosthetic strain. The results indicate that the functional status of human osteoblasts is determined by strain magnitude. The strain distribution in the proximal region of implanted femur should be improved for osseointegration.     

Mechanical Forces Induce Changes in VEGF and VEGFR-1/sFlt-1 Expression in Human Chondrocytes

Expression of the pro-angiogenic vascular endothelial growth factor (VEGF) stimulates angiogenesis and correlates with the progression of osteoarthritis. Mechanical joint loading seems to contribute to this cartilage pathology. Cyclic equibiaxial strains of 1% to 16% for 12 h, respectively, induced expression of VEGF in human chondrocytes dose- and frequency-dependently. Stretch-mediated VEGF induction was more prominent in the human chondrocyte cell line C-28/I2 than in primary articular chondrocytes. Twelve hours of 8% stretch induced VEGF expression to 175% of unstrained controls for at least 24 h post stretching, in promoter reporter and enzyme-linked immunosorbent assay (ELISA) studies. High affinity soluble VEGF-receptor, sVEGFR-1/sFlt-1 was less stretch-inducible than its ligand, VEGF-A, in these cells. ELISA assays demonstrated, for the first time, a stretch-mediated suppression of sVEGFR-1 secretion 24 h after stretching. Overall, strained chondrocytes activate their VEGF expression, but in contrast, strain appears to suppress the secretion of the major VEGF decoy receptor (sVEGFR-1/sFlt-1). The latter may deplete a biologically relevant feedback regulation to inhibit destructive angiogenesis in articular cartilage. Our data suggest that mechanical stretch can induce morphological changes in human chondrocytes in vitro. More importantly, it induces disturbed VEGF signaling, providing a molecular mechanism for a stress-induced increase in angiogenesis in cartilage pathologies.     

Downregulation of Runx2 by 1,25-Dihydroxyvitamin D3 Induces the Transdifferentiation of Osteoblasts to Adipocytes

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)₂D₃ enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)₂D₃ in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)₂D₃ enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)₂D₃ were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)₂D₃ induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts.     

Oxidative Stress-Induced Sirtuin1 Downregulation Correlates to HIF-1α, GLUT-1, and VEGF-A Upregulation in Th1 Autoimmune Hashimoto’s Thyroiditis

In Hashimoto’s thyroiditis (HT), oxidative stress (OS) is driven by Th1 cytokines’ response interfering with the normal function of thyrocytes. OS results from an imbalance between an excessive production of reactive oxygen species (ROS) and a lowering of antioxidant production. Moreover, OS has been shown to inhibit Sirtuin 1 (SIRT1), which is able to prevent hypoxia-inducible factor (HIF)-1α stabilization. The aims of this study were to determine the involvement of NADPH-oxidases (NOX), SIRT1, and HIF-1α in HT pathophysiology as well as the status of antioxidant proteins such as peroxiredoxin 1 (PRDX1), catalase, and superoxide dismutase 1 (SOD1). The protein expressions of NOX2, NOX4, antioxidant enzymes, SIRT1, and HIF-1α, as well as glucose transporter-1 (GLUT-1) and vascular endothelial growth factor A (VEGF-A), were analyzed by Western blot in primary cultures of human thyrocytes that were or were not incubated with Th1 cytokines. The same proteins were also analyzed by immunohistochemistry in thyroid samples from control and HT patients. In human thyrocytes incubated with Th1 cytokines, NOX4 expression was increased whereas antioxidants, such as PRDX1, catalase, and SOD1, were reduced. Th1 cytokines also induced a significant decrease of SIRT1 protein expression associated with an upregulation of HIF-1α, GLUT-1, and VEGF-A proteins. With the exception of PRDX1 and SOD1, similar results were obtained in HT thyroids. OS due to an increase of ROS produced by NOX4 and a loss of antioxidant defenses (PRDX1, catalase, SOD1) correlates to a reduction of SIRT1 and an upregulation of HIF 1α, GLUT-1, and VEGF-A. Our study placed SIRT1 as a key regulator of OS and we, therefore, believe it could be considered as a potential therapeutic target in HT.     

Molecular Actions of Ovarian Cancer G Protein-Coupled Receptor 1 Caused by Extracellular Acidification in Bone

Extracellular acidification occurs under physiologic and pathologic conditions, such as exercise, ischemia, and inflammation. It has been shown that acidosis has various adverse effects on bone. In recent years there has been increasing evidence which indicates that ovarian cancer G protein-coupled receptor 1 (OGR1) is a pH-sensing receptor and mediates a variety of extracellular acidification-induced actions on bone cells and other cell types. Recent studies have shown that OGR1 is involved in the regulation of osteoclast differentiation, survival, and function, as well as osteoblast differentiation and bone formation. Moreover, OGR1 also regulates acid-induced apoptosis of endplate chondrocytes in intervertebral discs. These observations demonstrate the importance of OGR1 in skeletal development and metabolism. Here, we provide an overview of OGR1 regulation ofosteoclasts, osteoblasts, and chondrocytes, and the molecular actions of OGR1 induced by extracellular acidification in the maintenance of bone health.     

Gelatin Methacryloyl–Riboflavin (GelMA–RF) Hydrogels for Bone Regeneration

Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been used in various biomedical fields. UV light is commonly used to photocrosslink such materials; however, its use has raised several biosafety concerns. We investigated the mechanical and biological properties of a visible-wavelength (VW)-light-crosslinked gelatin-based hydrogel to evaluate its viability as a scaffold for bone regeneration in bone-destructive disease treatment. Irgacure2959 or riboflavin was added as a photoinitiator to create GelMA solutions. GelMA solutions were poured into a mold and exposed to either UV or VW light. KUSA-A1 cell-laden GelMA hydrogels were crosslinked and then cultured. Mechanical characterization revealed that the stiffness range of GelMA–RF hydrogel was suitable for osteoblast differentiation. KUSA-A1 cells encapsulated in GelMA hydrogels photopolymerized with VW light displayed significantly higher cell viability than cells encapsulated in hydrogels photopolymerized with UV light. We also show that the expression of osteogenesis-related genes at a late stage of osteoblast differentiation in osteoblasts encapsulated in GelMA–RF hydrogel was markedly increased under osteoblast differentiation-inducing conditions. The GelMA–RF hydrogel served as an excellent scaffold for the encapsulation of osteoblasts. GelMA–RF hydrogel-encapsulated osteoblasts have the potential not only to help regenerate bone mass but also to treat complex bone defects associated with bone-destructive diseases such as periodontitis.     

Knee Osteoarthritis Progression Is Delayed in Silent Information Regulator 2 Ortholog 1 Knock-in Mice

Overexpression of silent information regulator 2 ortholog 1 (SIRT1) is associated with beneficial roles in aging-related diseases; however, the effects of SIRT1 overexpression on osteoarthritis (OA) progression have not yet been studied. The aim of this study was to investigate OA progression in SIRT1-KI mice using a mouse OA model. OA was induced via destabilization of the medial meniscus using 12-week-old SIRT1-KI and wild type (control) mice. OA progression was evaluated histologically based on the Osteoarthritis Research Society International (OARSI) score at 4, 8, 12, and 16 weeks after surgery. The production of SIRT1, type II collagen, MMP-13, ADAMTS-5, cleaved caspase 3, Poly (ADP-ribose) polymerase (PARP) p85, acetylated NF-κB p65, interleukin 1 beta (IL-1β), and IL-6 was examined via immunostaining. The OARSI scores were significantly lower in SIRT1-KI mice than those in control mice at 8, 12, and 16 weeks after surgery. The proportion of SIRT1 and type II collagen-positive-chondrocytes was significantly higher in SIRT1-KI mice than that in control mice. Moreover, the proportion of MMP-13-, ADAMTS-5-, cleaved caspase 3-, PARP p85-, acetylated NF-κB p65-, IL-1β-, and IL-6-positive chondrocytes was significantly lower in SIRT1-KI mice than that in control mice. The mechanically induced OA progression was delayed in SIRT1-KI mice compared to that in control mice. Therefore, overexpression of SIRT1 may represent a mechanism for delaying OA progression.     

HuR and TIA1/TIAL1 Are Involved in Regulation of Alternative Splicing of SIRT1 Pre-mRNA

SIRT1 is a pleiotropic protein that plays critical and multifunctional roles in metabolism, senescence, longevity, stress-responses, and cancer, and has become an important therapeutic target across a range of diseases. Recent research demonstrated that SIRT1 pre-mRNA undergoes alternative splicing to produce different isoforms, such as SIRT1 full-length and SIRT1-ΔExon8 variants. Previous studies revealed these SIRT1 mRNA splice variants convey different characteristics and functions to the protein, which may in turn explain the multifunctional roles of SIRT1. However, the mechanisms underlying the regulation of SIRT1 alternative splicing remain to be elucidated. Our objective is to search for new pathways that regulate of SIRT1 alternative splicing. Here we describe experiments showing that HuR and TIA1/TIAL1, two kinds of RNA-binding proteins, were involved in the regulation of alternative splicing of SIRT1 pre-mRNA under normal and stress circumstances: HuR increased SIRT1-ΔExon8 by promoting SIRT1 exon 8 exclusion, whereas TIA1/TIAL1 inhibition of the exon 8 exclusion led to a decrease in SIRT1-ΔExon8 mRNA levels. This study provides novel insight into how the alternative splicing of SIRT1 pre-mRNA is regulated, which has fundamental implications for understanding the critical and multifunctional roles of SIRT1.     

Transient Receptor Potential Ankyrin 1 (TRPA1) Is Involved in Upregulating Interleukin-6 Expression in Osteoarthritic Chondrocyte Models

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-bound ion channel found in neurons, where it mediates nociception and neurogenic inflammation. Recently, we have discovered that TRPA1 is also expressed in human osteoarthritic (OA) chondrocytes and downregulated by the anti-inflammatory drugs aurothiomalate and dexamethasone. We have also shown TRPA1 to mediate inflammation, pain, and cartilage degeneration in experimental osteoarthritis. In this study, we investigated the role of TRPA1 in joint inflammation, focusing on the pro-inflammatory cytokine interleukin-6 (IL-6). We utilized cartilage/chondrocytes from wild-type (WT) and TRPA1 knockout (KO) mice, along with primary chondrocytes from OA patients. The results show that TRPA1 regulates the synthesis of the OA-driving inflammatory cytokine IL-6 in chondrocytes. IL-6 was highly expressed in WT chondrocytes, and its expression, along with the expression of IL-6 family cytokines leukemia inhibitory factor (LIF) and IL-11, were significantly downregulated by TRPA1 deficiency. Furthermore, treatment with the TRPA1 antagonist significantly downregulated the expression of IL-6 in chondrocytes from WT mice and OA patients. The results suggest that TRPA1 is involved in the upregulation of IL-6 production in chondrocytes. These findings together with previous results on the expression and functions of TRPA1 in cellular and animal models point to the role of TRPA1 as a potential mediator and novel drug target in osteoarthritis.     

The Therapeutic Potential of Zinc-Alpha2-Glycoprotein (AZGP1) in Fibrotic Kidney Disease

Chronic kidney disease (CKD) is characterized by a long-term loss of kidney function and, in most cases, by progressive fibrosis. Zinc-alpha2-glycoprotein (AZGP1) is a secreted protein, which is expressed in many different tissues and has been associated with a variety of functions. In a previous study, we have shown in cell culture and in AZGP1 deficient mice that AZGP1 has protective anti-fibrotic effects. In the present study, we tested the therapeutic potential of an experimental increase in AZGP1 using two different strategies. (1) C57Bl/6J mice were treated systemically with recombinant AZGP1, and (2) a transgenic mouse strain was generated to overexpress AZGP1 conditionally in proximal tubular cells. Mice underwent unilateral uretic obstruction as a pro-fibrotic kidney stress model, and kidneys were examined after 14 days. Recombinant AZGP1 treatment was accompanied by better preservation of tubular integrity, reduced collagen deposition, and lower expression of injury and fibrosis markers. Weaker but similar tendencies were observed in transgenic AZGP1 overexpressing mice. Higher AZGP1 levels led to a significant reduction in stress-induced accumulation of tubular lipid droplets, which was paralleled by improved expression of key players in lipid metabolism and fatty acid oxidation. Together these data show beneficial effects of elevated AZGP1 levels in fibrotic kidney disease and highlight a novel link to tubular cell lipid metabolism, which might open up new opportunities for CKD treatment.     

Ferulic Acid,Pterostilbene, and Tyrosol Protect the Heart from ER-Stress-Induced Injury by Activating SIRT1-Dependent Deacetylation of eIF2α

Disturbances in Endoplasmic Reticulum (ER) homeostasis induce ER stress, which has been involved in the development and progression of various heart diseases, including arrhythmias, cardiac hypertrophy, ischemic heart diseases, dilated cardiomyopathy, and heart failure. A mild-to-moderate ER stress is considered beneficial and adaptative for heart functioning by engaging the pro-survival unfolded protein response (UPR) to restore normal ER function. By contrast, a severe or prolonged ER stress is detrimental by promoting cardiomyocyte apoptosis through hyperactivation of the UPR pathways. Previously, we have demonstrated that the NAD⁺-dependent deacetylase SIRT1 is cardioprotective in response to severe ER stress by regulating the PERK pathway of the UPR, suggesting that activation of SIRT1 could protect against ER-stress-induced cardiac damage. The purpose of this study was to identify natural molecules able to alleviate ER stress and inhibit cardiomyocyte cell death through SIRT1 activation. Several phenolic compounds, abundant in vegetables, fruits, cereals, wine, and tea, were reported to stimulate the deacetylase activity of SIRT1. Here, we evaluated the cardioprotective effect of ten of these phenolic compounds against severe ER stress using cardiomyoblast cells and mice. Among the molecules tested, we showed that ferulic acid, pterostilbene, and tyrosol significantly protect cardiomyocytes and mice heart from cardiac alterations induced by severe ER stress. By studying the mechanisms involved, we showed that the activation of the PERK/eIF2α/ATF4/CHOP pathway of the UPR was reduced by ferulic acid, pterostilbene, and tyrosol under ER stress conditions, leading to a reduction in cardiomyocyte apoptosis. The protection afforded by these phenolic compounds was not directly related to their antioxidant activity but rather to their ability to increase SIRT1-mediated deacetylation of eIF2α. Taken together, our results suggest that ferulic acid, pterostilbene, and tyrosol are promising molecules to activate SIRT1 to protect the heart from the adverse effects of ER stress.     

The mTORC2 Regulator Homer1 Modulates Protein Levels and Sub-Cellular Localization of the CaSR in Osteoblast-Lineage Cells

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including β-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR−/− mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR−/− mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.     

The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization

KCNQ (K_V7) channels are voltage-gated potassium (K_V) channels, and the function of K_V7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of K_V channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that K_V7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional K_V7 currents were detected in MG-63 cells. Inhibition of K_V7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of K_V7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of K_V7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that K_V7.3 could be a novel regulator in osteoblast differentiation.