Morten A. Karsdal

The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation.

Bone resorption may generate collagen fragments such as ICTP and CTX, which can be quantified in serum and/or urine by using specific immunoassays, and which are used as clinical markers. However, the relative abundance of ICTP and CTX varies according to the type of bone pathology, suggesting that these two fragments are generated through distinct collagenolytic pathways. In this study, we analyzed the release of ICTP and CTX from bone collagen by the proteinases reported to play a role in the solubilization of bone matrix. Cathepsin K released large amounts of CTX, but did not allow a detectable release of ICTP. Conversely, the matrix metalloproteinases (MMPs) MMP‐2, ‐9, ‐13, or ‐14 released ICTP, but did not allow a detectable release of CTX. Next we analyzed the release of ICTP and CTX from bone explants cultured in the presence of well‐established inhibitors of these proteinases and of matrix solubilization. An inhibitor of cysteine proteinases including cathepsin K, inhibited the release of CTX, but not the release of ICTP. MMP inhibitors inhibited the release of ICTP, but also that of CTX, in agreement with the putative role of MMPs in the initiation of bone resorption in addition to matrix solubilization. Similarly the treatment of mice bearing bone metastasis with an MMP inhibitor led to a significant reduction of serum ICTP and CTX, and osteolytic lesions. We conclude that the generation of ICTP and CTX depends on different collagenolytic pathways. This finding may explain why these two markers may discriminate between different bone pathologies.

Are nonresorbing osteoclasts sources of bone anabolic activity

Some osteopetrotic mutations lead to low resorption, increased numbers of osteoclasts, and increased bone formation, whereas other osteopetrotic mutations lead to low resorption, low numbers of osteoclasts, and decreased bone formation. Elaborating on these findings, we discuss the possibility that osteoclasts are the source of anabolic signals for osteoblasts. In normal healthy individuals, bone formation is coupled to bone resorption in a tight equilibrium. When this delicate balance is disturbed, the net result is pathological situations, such as osteopetrosis or osteoporosis. Human osteopetrosis, caused by mutations in proteins involved in the acidification of the resorption lacuna (ClC‐7 or the a3‐V‐ATPase), is characterized by decreased resorption in face of normal or even increased bone formation. Mouse mutations leading to ablation of osteoclasts (e.g., loss of macrophage‐colony stimulating factor [M‐CSF] or c‐fos) lead to secondary negative effects on bone formation, in contrast to mutations where bone resorption is abrogated with sustained osteoclast numbers, such as the c‐src mice. These data indicate a central role for osteoclasts, and not necessarily their resorptive activity, in the control of bone formation. In this review, we consider the balance between bone resorption and bone formation, reviewing novel data that have shown that this principle is more complex than originally thought. We highlight the distinct possibility that osteoclast function can be divided into two more or less separate functions, namely bone resorption and stimulation of bone formation. Finally, we describe the likely possibility that bone resorption can be attenuated pharmacologically without the undesirable reduction in bone formation.

Local communication on and within bone controls bone remodeling

Bone remodeling is required for healthy calcium homeostasis and for repair of damage occurring with stress and age. Osteoclasts resorb bone and osteoblasts form bone. These processes normally occur in a tightly regulated sequence of events, where the amount of formed bone equals the amount of resorbed bone, thereby restoring the removed bone completely. Osteocytes are the third cell type playing an essential role in bone turnover. They appear to regulate activation of bone remodeling, and they exert both positive and negative regulation on both osteoclasts and osteoblasts. In this review, we consider the intricate communication between these bone cells in relation to bone remodeling, reviewing novel data from patients with mutations rendering different cell populations inactive, which have shown that these interactions are more complex than originally thought. We highlight the high probability that a detailed understanding of these processes will aid in the development of novel treatments for bone metabolic disorders, i.e. we discuss the possibility that bone resorption can be attenuated pharmacologically without a secondary reduction in bone formation.

Proteinases in bone resorption: obvious and less obvious roles

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.

Acidification of the Osteoclastic Resorption Compartment Provides Insight into the Coupling of Bone Formation to Bone Resorption

Patients with defective osteoclastic acidification have increased numbers of osteoclasts, with decreased resorption, but bone formation that remains unchanged. We demonstrate that osteoclast survival is increased when acidification is impaired, and that impairment of acidification results in inhibition of bone resorption without inhibition of bone formation. We investigated the role of acidification in human osteoclastic resorption and life span in vitro using inhibitors of chloride channels (NS5818/NS3696), the proton pump (bafilomycin) and cathepsin K. We found that bafilomycin and NS5818 dose dependently inhibited acidification of the osteoclastic resorption compartment and bone resorption. Inhibition of bone resorption by inhibition of acidification, but not cathepsin K inhibition, augmented osteoclast survival, which resulted in a 150 to 300% increase in osteoclasts compared to controls. We investigated the effect of inhibition of osteoclastic acidification in vivo by using the rat ovariectomy model with twice daily oral dosing of NS3696 at 50 mg/kg for 6 weeks. We observed a 60% decrease in resorption (DPYR), increased tartrate-resistant acid phosphatase levels, and no effect on bone formation evaluated by osteocalcin. We speculate that attenuated acidification inhibits dissolution of the inorganic phase of bone and results in an increased number of nonresorbing osteoclasts that are responsible for the coupling to normal bone formation. Thus, we suggest that acidification is essential for normal bone remodeling and that attenuated acidification leads to uncoupling with decreased bone resorption and unaffected bone formation.

The Chloride Channel Inhibitor NS3736 Prevents Bone Resorption in Ovariectomized Rats Without Changing Bone Formation

Chloride channel activity is essential for osteoclast function. Consequently, inhibition of the osteoclastic chloride channel should prevent bone resorption. Accordingly, we tested a chloride channel inhibitor on bone turnover and found that it inhibits bone resorption without affecting bone formation. This study indicates that chloride channel inhibitors are highly promising for treatment of osteoporosis.

Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity

IntroductionPhysiological and pathophysiological cartilage turnover may coexist in articular cartilage. The distinct enzymatic processes leading to irreversible cartilage damage, compared with those needed for continuous self-repair and regeneration, remain to be identified. We investigated the capacity of repair of chondrocytes by analyzing their ability to initiate an anabolic response subsequent to three different levels of catabolic stimulation.MethodsCartilage degradation was induced by oncostatin M and tumour necrosis factor in articular cartilage explants for 7, 11, or 17 days. The catabolic period was followed by 2 weeks of anabolic stimulation (insulin growth factor-I). Cartilage formation was assessed by collagen type II formation (PIINP). Cartilage degradation was measured by matrix metalloproteinase (MMP) mediated type II collagen degradation (CTX-II), and MMP and aggrecanase mediated aggrecan degradation by detecting the 342FFGVG and 374ARGSV neoepitopes. Proteoglycan turnover, content, and localization were assessed by Alcian blue.ResultsCatabolic stimulation resulted in increased levels of cartilage degradation, with maximal levels of 374ARGSV (20-fold induction), CTX-II (150-fold induction), and 342FFGVG (30-fold induction) (P < 0.01). Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages. Anabolic treatment increased proteoglycan content at all time points (maximally, 250%; P < 0.001). By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point. In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.ConclusionCartilage degradation was completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation. After induction of MMP-mediated aggrecan and collagen type II degradation, the chondrocytes had impaired repair capacity.

Transforming Growth Factor-β Controls Human Osteoclastogenesis through the p38 MAPK and Regulation of RANK Expression

Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-β (TGF-β) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-β in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-β on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-β was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-β during the initial culture period (days 1–7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-β was present during the entire culture period (days 1–21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-β was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-β, we examined RANK expression and p38 MAPK activation. We found the TGF-β strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-β to monocytes down-regulated RANK expression. The current results suggest that TGF-β promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-β abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling.

The future of blood-based biomarkers for Alzheimer's disease

Treatment of Alzheimers disease (AD) is significantly hampered by the lack of easily accessible biomarkers that can detect disease presence and predict disease risk reliably. Fluid biomarkers of AD currently provide indications of disease stage; however, they are not robust predictors of disease progression or treatment response, and most are measured in cerebrospinal fluid, which limits their applicability. With these aspects in mind, the aim of this article is to underscore the concerted efforts of the Blood‐Based Biomarker Interest Group, an international working group of experts in the field. The points addressed include: (1) the major challenges in the development of blood‐based biomarkers of AD, including patient heterogeneity, inclusion of the “right” control population, and the blood–brain barrier; (2) the need for a clear definition of the purpose of the individual markers (e.g., prognostic, diagnostic, or monitoring therapeutic efficacy); (3) a critical evaluation of the ongoing biomarker approaches; and (4) highlighting the need for standardization of preanalytical variables and analytical methodologies used by the field.

An update on biomarkers of bone turnover and their utility in biomedical research and clinical practice

Abstract:Background:Maintenance of the structural and functional integrity of the skeleton is a critical function of a continuous remodeling driven by highly associated processes of bone resorption and synthetic activities driven by osteoclasts and osteoblasts, respectively. Acceleration of bone turnover, accompanied with a disruption of the coupling between these cellular activities, plays an established role in the pathogenesis of metabolic bone diseases, such as osteoporosis. During the past decades, major efforts have been dedicated to the development and clinical assessment of biochemical markers that can reflect the rate of bone turnover. Numerous studies have provided evidence that serum levels or urinary excretion of these biomarkers correlate with the rate of bone loss and fracture risk, proving them as useful tools for improving identification of high-risk patients.Objective:The aim of the present review is to give an update on biomarkers of bone turnover and give an overview of their applications in epidemiological and clinical research.Discussion:Special attention is given to their utility in clinical trials testing the efficacy of drugs for the treatment of osteoporosis and how they supplement bone mass measurements. Recent evidence suggests that biochemical markers may provide information on bone age that may have indirectly relates to bone quality; the latter is receiving increasing attention. A more targeted use of biomarkers could further optimize identification of high-risk patients, the process of drug discovery, and monitoring of the efficacy of osteoporosis treatment in clinical settings.