Jonathan D. Holz

Osthole Stimulates Osteoblast Differentiation and Bone Formation by Activation of β-Catenin-BMP Signaling

Osteoporosis is defined as reduced bone mineral density with a high risk of fragile fracture. Current available treatment regimens include antiresorptive drugs such as estrogen receptor analogues and bisphosphates and anabolic agents such as parathyroid hormone (PTH). However, neither option is completely satisfactory because of adverse effects. It is thus highly desirable to identify novel anabolic agents to improve future osteoporosis treatment. Osthole, a coumarin‐like derivative extracted from Chinese herbs, has been shown to stimulate osteoblast proliferation and differentiation, but its effect on bone formation in vivo and underlying mechanism remain unknown. In this study, we found that local injection of Osthole significantly increased new bone formation on the surface of mouse calvaria. Ovariectomy caused evident bone loss in rats, whereas Osthole largely prevented such loss, as shown by improved bone microarchitecture, histomorphometric parameters, and biomechanical properties. In vitro studies demonstrated that Osthole activated Wnt/β‐catenin signaling, increased Bmp2 expression, and stimulated osteoblast differentiation. Targeted deletion of the β‐catenin and Bmp2 genes abolished the stimulatory effect of Osthole on osteoblast differentiation. Since deletion of the Bmp2 gene did not affect Osthole‐induced β‐catenin expression and the deletion of the β‐catenin gene inhibited Osthole‐regulated Bmp2 expression in osteoblasts, we propose that Osthole acts through β‐catenin–BMP signaling to promote osteoblast differentiation. Our findings demonstrate that Osthole could be a potential anabolic agent to stimulate bone formation and prevent estrogen deficiency–induced bone loss.

Environmental Toxicants May Modulate Osteoblast Differentiation by a Mechanism Involving the Aryl Hydrocarbon Receptor

The AHR mediates many of the toxicological effects of aromatic hydrocarbons. We show that AHR expression in osteoblasts parallels the induction of early bone‐specific genes involved in maturation. The AHR may not only mediate the effects of toxicants, but with an as yet unidentified ligand, be involved in the differentiation pathways of osteoblasts.

Icariin Augments Bone Formation and Reverses the Phenotypes of Osteoprotegerin-Deficient Mice through the Activation of Wnt/β-Catenin-BMP Signaling

Icariin has been mostly reported to enhance bone fracture healing and treat postmenopausal osteoporosis in ovariectomized animal model. As another novel animal model of osteoporosis, there is few publication about the effect of Icariin on osteoprotegerin-deficient mice. Therefore, the goal of this study is to find the effect on bone formation and underlying mechanisms of Icariin in osteoprotegerin (OPG) knockout (KO) mice. We found that Icariin significantly stimulated new bone formation after local injection over the surface of calvaria at the dose of 5 mg/kg per day. With this dose, Icariin was also capable of significantly reversing OPG-deficient-induced bone loss and bone strength reduction. Real-time PCR analysis showed that Icariin significantly upregulated the expression of BMP2, BMP4, RUNX2, OC, Wnt1, and Wnt3a in OPG KO mice. Icariin also significantly increased the expression of AXIN2, DKK1, TCF1, and LEF1, which are the direct target genes of β-catenin signaling. The in vitro studies showed that Icariin induced osteoblast differentiation through the activation of Wnt/β-catenin-BMP signaling by in vitro deletion of the β-catenin gene using β-cateninfx/fx mice. Together, our findings demonstrate that Icariin significantly reverses the phenotypes of OPG-deficient mice through the activation of Wnt/β-catenin-BMP signaling.

Heavy Metal Ion Regulation of Gene Expression: MECHANISMS BY WHICH LEAD INHIBITS OSTEOBLASTIC BONE-FORMING ACTIVITY THROUGH MODULATION OF THE Wnt/β-CATENIN SIGNALING PATHWAY.

Background: Mechanistic information regarding how lead exposure decreases bone mass does not exist in the literature. Results: Pb exposure decreases bone formation and inhibits Wnt signaling; this effect is mitigated by the deletion of sclerostin. Conclusion: Pb inhibition of the Wnt pathway impedes osteoblast activity and is mediated by blockade of the TGFβ/Smad3 pathway. Significance: This study provides insight regarding Pb suppression of bone-forming activity. Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/β-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the β-catenin pathway using a pharmacological inhibitor of GSK-3β ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFβ canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/β-catenin pathway.

Gene Expression Profile of Steroid-induced Necrosis of Femoral Head of Rats

The key to treating steroid-induced necrosis of femoral heads (SINFH) is early diagnosis. Dramatic improvements in diagnosis could be made if the pathogenesis of SINFH was more fully understood; however, the underlying mechanism of this disease is currently unknown. To explore the potential mechanism of SINFH, we performed gene array analysis on a rat model of the disease and compare the expression profile with that of normal rats. A quantitative RT-PCR and immunohistochemistry (IHC) assays were used to confirm the microarray results. Compared to the control group, 190 genes in the experimental group were differentially expressed, with 52 up-regulated and 138 down-regulated. Of these genes, 102 are known (deposited in GenBank), while 88 of them are unknown. The known genes can be divided into several families according to their biological functions, such as oxidative stress, apoptosis, signal transduction, angiogenesis, extracellular matrix, lipid metabolism, and transcription related genes. The results of quantitative RT-PCR and IHC were consistent with gene chip results. Our findings indicate that many genes involved in diverse signaling pathways were differentially expressed between SINFH rats and normal rats. Furthermore, our findings suggest that the development of SINFH is a complicated and dynamic process affected by multiple factors and signaling pathways and regulated by various genes.

Lead induces an osteoarthritis‐like phenotype in articular chondrocytes through disruption of TGF‐β signaling

Lead remains a significant environmental toxin, and we believe we may have identified a novel target of lead toxicity in articular chondrocytes. These cells are responsible for the maintenance of joint matrix, and do so under the regulation of TGF‐β signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF‐β signaling. These experiments examine the effects of lead exposure in vivo and in vitro at biologically relevant levels, from 1 nM to 10 µM on viability, collagen levels, matrix degrading enzyme activity, TGF‐β signaling, and articular surface morphology. Our results indicate that viability was unchanged at levels ≤100 µM Pb, but low and high level lead in vivo exposure resulted in fibrillation and degeneration of the articular surface. Lead treatment also decreased levels of type II collagen and increased type X collagen, in vivo and in vitro. Additionally, MMP13 activity increased in a dose‐dependent manner. Active caspase 3 and 8 were dose‐dependently elevated, and treatment with 10 µM Pb resulted in increases of 30% and 500%, respectively. Increasing lead treatment resulted in a corresponding reduction in TGF‐β reporter activity, with a 95% reduction at 10µM. Levels of phosphoSmad2 and 3 were suppressed in vitro and in vivo and lead dose‐dependently increased Smurf2. These changes closely parallel those seen in osteoarthritis. Over time this phenotypic shift could compromise maintenance of the joint matrix.

Changes of Cervical Dorsal Root Ganglia Induced by Compression Injury and Decompression Procedure: A Novel Rat Model of Cervical Radiculoneuropathy

Our study aimed to establish a model of compression injury of cervical dorsal root ganglia (DRG) in the rat and to investigate the pathological changes following compression injury and decompression procedures. Thirty rats were divided into three groups: control group receiving sham surgery, compression group undergoing surgery to place a micro-silica gel on C6 DRG, and decompression group with subsequent decompression procedure. The samples harvested from the different groups were examined with light microscopy, ultrastructural analysis, and horseradish peroxidase (HRP) retrograde tracing techniques. Apoptosis of DRG neurons was demonstrated with TUNEL staining. Changes in PGE2 and PLA2 in DRG neurons were detected with enzyme-linked immunosorbent assay (ELISA). Local expression of vascular endothelial growth factor (VEGF) was monitored with immunohistochemistry. DRG neurons in the compression group became swollen with vacuolar changes in cytoplasm. Decompression procedure partially ameliorated the resultant compression pathology. Ultrastructural examination showed a large number of swollen vacuoles, demyelinated nerve root fibers, absence of Schwann cells, and proliferation in the surrounding connective tissues in the compression group. Compared to the control group, the compression group showed a significant decrease in the number of the HRP-labeled cells and a significant increase in levels of PGE2 and PLA2, in the expression of VEGF protein, and in the number of apoptotic DRG neurons. These findings demonstrate that compression results in local inflammation, followed by increased apoptosis and upregulation of VEGF. We conclude that such a model provides a tool to study the pathogenesis and treatment of cervical radiculoneuropathy.

CHAPTER 15:Lead Exposure and Osteoporosis: Mechanisms and Clinical Manifestations

The heavy metal lead remains a dangerous toxin in our environment, adversely affecting the functioning of virtually all tissues. Although it has been a lesser investigated organ system, the skeleton turns out to be a key target. Leads effects on bone are multifactorial and create a series of detrimental responses that can i) cause a serious pathological condition, ii) mask its diagnosis and iii) inhibit the bodys ability to heal the condition. Specifically, lead is now known to inhibit osteoblastic bone formation, interfere with the detection of low bone mass by X-ray based techniques and compromise skeletal fracture healing.The level of lead in our environment remains high. It does not degrade with time, and lead continues to have easy access to the body through airborne, food and water routes. Unfortunately, lead accumulates in bone. Its ionic radius is similar to that of calcium, allowing incorporation into the hydroxyapatite crystal structure of bone. It has been estimated that the half-life of lead in the skeleton is of the order of decades. It is not uncommon to find individuals with low blood levels and high bone levels of lead.It appears that lead has direct effects on bone cell signaling pathway intermediates, as well as key kinases. By interfering in the bone formation process it tends to uncouple the remodeling process, thereby shifting the balance of maintaining skeletal bone mass toward chronic loss, resulting in an osteoporotic-like syndrome. This is most clearly a problem in older adults.