Old age-associated phenotypic screening for Alzheimer's disease drug candidates identifies sterubin as a potent neuroprotective compound from Yerba santa

Alzheimer's disease (AD) is the most frequent age-associated dementia with no treatments that can prevent or slow its progression. Since age is by far the major risk factor for AD, there is a strong rationale for an alternative approach to drug discovery based upon the biology of aging. Phenotypic screening assays that reflect multiple, age-associated neurotoxicity pathways rather than single molecular targets can identify compounds that have therapeutic efficacy by targeting aspects of aging that contribute to AD pathology. And, while the suitability of any single assay can be questioned, a combination of assays can make reliable predictions about the neuroprotective effects of compounds in vivo. Therefore, our lab has developed a combination of phenotypic screening assays that are ideally suited not only to identify novel neuroprotective compounds for the treatment of AD but also their target pathways, thereby potentially providing new therapeutic targets for disease treatment. Using these assays, we screened a large library of extracts from plants with identified pharmacological uses. Analysis of one of these extracts from the plant Yerba santa (Eriodictyon californicum) identified the flavanone sterubin as the active component and further studies showed it to be a potent neuroprotective and anti-inflammatory compound.

Introduction
Since the 1990's, the combination of molecular and structural biology, combinatorial chemistry and high throughput screening has dominated drug discovery [1]. This approach provides a rapid process for the discovery of drug candidates with high selectivity and affinity for a specific molecular target. However, it has not produced the successes that were initially expected, especially with respect to complex neurodegenerative diseases such as Alzheimer's disease (AD).
Prior to the development of this target-based drug discovery paradigm, new drugs were discovered by evaluating chemicals against observable characteristics or phenotypes in biological systems such as cells or animals. This method is known as phenotypic screening. While this approach has fallen out of favor with the pharmaceutical industry, surprisingly a recent study showed that it still continues to be more successful than target-based approaches for the identification of first in class small molecule drugs [2]. It has been argued that this is because target-based discovery is based on a priori assumptions that do not take into account the complexities of biological systems [2,3]. Moreover, phenotypic screening against human disease models can lead to the identification of new, disease-related molecular pathways and targets.
The ideal phenotypic drug screening paradigm would employ the ultimate end user-humans; and this is how most of the natural productbased, first in class drugs were originally discovered. However, for obvious reasons, this strategy is no longer ethically viable. Laboratory animals, primarily disease models in mice, are currently used for preclinical testing. However, using them for the initial screening of drug candidates is impractical due to cost and time constraints as well as the drive to reduce animal use in research. A reasonable alternative is to create cell-based assays based on toxicity pathways relevant to ageassociated neurodegeneration and use these assays to identify novel drug candidates. In this way, the screening paradigms have disease relevance, reproducibility and reasonable throughput.
Many arguments can be made against the relevance of any single cellular screening assay, based on the cell type or the nature of the toxic insult. Thus, to account for individual weaknesses, our phenotypic screening approach combines multiple assays. This enables the identification of potent, disease-modifying compounds for preclinical testing in animal models of neurodegenerative diseases. In general, these assays involve primary neurons, neuron-like cell lines or microglial cell lines that are subjected to toxic insults that have been observed to occur in the aging brain and to a larger extent in AD.
In this report, we describe the use of these assays to screen a commercial library of extracts from plants with identified pharmacological uses and the identification of a previously uncharacterized neuroprotective flavonoid. All plant extracts were first tested in the oxytosis assay in HT22 mouse hippocampal nerve cells. Extracts that were positive in this assay were then screened in additional assays including: protection against energy depletion in HT22 hippocampal nerve cells, intracellular amyloid toxicity in MC65 human nerve cells, inhibition of inflammation mediated by microglial activation using BV-2 mouse microglial cells and differentiation of rat PC12 cells. These assays reflect multiple, age-associated neurotoxicity/survival pathways directly relevant to AD, such as increased oxidative stress and glutathione (GSH) depletion, reduced energy metabolism, accumulation of misfolded, aggregated proteins, loss of neurotrophic support and inflammation [3]. In addition, these particular models were selected to provide a replicable, cost-and time-effective screening approach.

Materials and methods
All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. The plant extract library was obtained from Caithness Biotechnologies (Leicester, UK). Eriodictyol and homoeriodictyol were purchased from Indofine Chemical Company (Hillsborough, NJ, USA). Sterubin was a gift from Jakob Ley at Symrise AG (Holzminden, Germany).

Oxytosis (HT22 cells)
This assay, also called oxidative glutamate toxicity, tests the ability of compounds to rescue cells from oxidative stress-induced programmed cell death caused by GSH depletion after treatment with glutamate [4]. A reduction in GSH is seen in the aging brain and is accelerated in AD [5]. The depletion of GSH from cells leads to lipoxygenase activation, reactive oxygen species production and calcium influx which initiates a form of programmed cell death with features similar to those implicated in the nerve cell damage seen in AD [6]. Because of the generality of the toxicity pathway in oxytosis and its mechanistic association with aging and AD [7,8], it is used as our primary screen. In this assay, 5 × 10 3 HT22 mouse hippocampal nerve cells, grown in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logan, UT, USA), were plated in 96 well plates. After 24 h of culture, the medium was exchanged with fresh medium and 5 mM glutamate and the indicated concentrations/dilutions of extracts/fractions/compounds were added. After 24 h of treatment, viability was measured by the 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described [9]. Results were confirmed by visual inspection of the wells.

Anti-inflammatory activity (BV-2 cells)
Inflammation is a major feature of AD [10]. Microglia are the resident immune cell population of the CNS and activated, pro-inflammatory microglia are implicated in the pathogenesis of AD [10]. Activated microglia produce a wide array of pro-inflammatory and cytotoxic factors including cytokines, free radicals, excitatory neurotransmitters and eicosanoids that may work in concert to promote neurodegeneration [10]. Thus, inhibiting the generation of activated pro-inflammatory microglia is another important therapeutic target for AD. Briefly, mouse BV2 microglial cells were grown in low glucose DMEM supplemented with 10% FCS. For the assay the cells were plated at 5 × 10 5 cells in 35 mm tissue culture dishes. After growth overnight, the cells were treated with 50 µg/ml bacterial lipopolysaccharide (LPS) alone or in the presence of the extracts/compounds. After 24 h the medium was removed, spun briefly to remove floating cells and 100 µl assayed for nitrite using 100 µl of the Griess Reagent in a 96 well plate. After incubation for 10 min at room temperature the absorbance at 550 nm was read on a microplate reader, as described previously [11]. The levels of IL-6, IL1β and TNF-α in the supernatants were determined using ELISAs (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instruction.

Neurotrophic activity (PC12 cell differentiation)
Connections between nerve cells are altered in AD. Thus, compounds that can promote the regeneration of these connections might be of particular benefit, thereby promoting the recovery of higher neuronal function. As a model for this property, we used neurite outgrowth in rat PC12 cells, a well-studied model system of neuronal differentiation. In response to neurotrophic factors such as nerve growth factor (NGF), PC12 cells undergo a series of physiological changes culminating in a phenotype resembling that of sympathetic neurons [12]. In this assay, PC12 cells (originally obtained from Greene [13]) grown in high-glucose DMEM supplemented with 10% FCS and 5% horse serum were plated in 35 mm tissue culture dishes. After 3 days of growth, the medium was replaced with serum-free N2 medium (Invitrogen) and the cells were treated with the extracts/compounds. After 24 h the cells were scored for the presence of neurites. PC12 cells produce neurites much more rapidly when treated in N2 medium than when treated in regular growth medium. For each treatment, 100 cells in each of three separate fields were counted. Cells were scored positive if one or more neurites longer than one cell body diameter in length were observed [14].

Protection against energy loss (HT22 cells)
A breakdown in neuronal energy production leading to adenosine triphosphate (ATP) loss is associated with nerve cell damage and death in AD [15]. In order to induce ATP loss, we used the compound iodoacetic acid (IAA) in combination with the HT22 hippocampal nerve cells. IAA has been used in a number of other studies to cause a rapid loss of ATP [16]. HT22 cells were seeded onto 96 well plates as described in the oxytosis assay. The medium was exchanged 24 h later with fresh medium and the cells were treated with 20 µM IAA alone (which results in 90-95% cell death) or in the presence of extracts/ compounds. After 2 h the medium in each well was aspirated and replaced with fresh medium without IAA but containing the extracts/ compounds. After 24 h of treatment, viability was measured by the MTT assay. Results were confirmed by visual inspection of the wells.

Intracellular abeta toxicity (MC65 cells)
Accumulation of intracellular amyloid beta peptide (Aβ) is considered by many as being a primary toxic event in AD [17]. The human nerve cell line MC65 conditionally expresses the C99 fragment of the amyloid precursor protein (APP) leading to the accumulation of intracellular Aβ. The MC65 cells are routinely grown in the presence of tetracycline and, following its removal, the expression of C99 is induced and the cells die within 4 days because of the accumulation of intracellular, toxic protein aggregates [18,19]. Briefly, MC65 cells were regularly grown in high glucose DMEM supplemented with 10% FCS. For the assay, cells were dissociated and plated at 4 × 10 5 cells per 35 mm tissue culture dishes in Opti-minimal essential media (Opti-MEM, Invitrogen) in the presence (no induction) or absence (APP-C99 induced) of 1 μg/ml tetracycline in the presence or absence of the indicated extracts/compounds. At day 4, the control cells in the absence of tetracycline were dead, and cell viability was determined by the MTT assay, as previously described [19].

HPLC fractionation
Initially, the plant extract (1 µl of original DMSO extract) was diluted with 150 µl of 50% aqueous acetonitrile and injected onto a Vydac C-18 column (4.6 × 250 mm). A linear gradient was run over 30 min, beginning at 9% acetonitrile in 0.1% trifluoroacetic acid and increasing to 90% acetonitrile with a flow rate of 1 ml/min. Absorbance was monitored at 330 nm. One milliliter fractions were collected automatically for the entire gradient. Fractions were dried in vacuo employing a Savant Speed-Vac concentrator and fractions were re-dissolved in DMSO before testing in the oxytosis assay described above. Once the active fraction was identified, a shallower HPLC gradient was employed (40 min, beginning at 18% acetonitrile in 0.1% trifluoroacetic acid increasing to 54% acetonitrile with a flow rate of 1 ml/min) and fractions surrounding the active peak were collected manually based on absorbance at 330 nm. An aliquot of these fractions was dried in vacuo and assayed in the oxytosis assay. The active fraction was then subjected to mass spectrometric analysis.

Mass spectrometry
The fraction eluting at 19.4 min under the HPLC conditions described above was diluted 1:100 with 1% formic acid in methanol/ water. The analyte was directly infused into a ThermoFisher Q Exactive mass spectrometer (Waltham, MA, USA) via a micro-flow source. A major signal was detected at m/z = 303.09 (singly charged). The signal was isolated and subjected to HCD fragmentation at a normalized collision energy setting of 45. MS/MS spectra were recorded and averaged for 2 min. After the analysis of the natural compound, spectra were also recorded for the synthetic compounds (homoeriodictyol, hesperitin, and sterubin), all of which possess a predicted precursor mass of MH + = 303.09. Only the MS/MS spectrum of pure sterubin exhibited a match with that of the isolated natural product.

Measurement of total glutathione (tGSH)
For measurement of tGSH, 3 × 10 5 HT22 cells were plated in 60 mm dishes. After 24 h of culture, the medium was exchanged with fresh medium and the indicated concentrations of glutamate and the compounds were added. The cells were treated for 2-24 h and then scraped into ice-cold PBS and 10% sulfosalicylic acid was added at a final concentration of 3.3%. GSH was determined by the recycling assay based on the reduction of 5,5-dithiobis(2-nitrobenzoic acid) with glutathione reductase and NADPH [20] and normalized to protein recovered from the acid-precipitated pellet by treatment with 0.2 N NaOH at 37°C overnight and measured by the bicinchoninic acid assay (Pierce, Rockford, IL, USA).

Reactive oxygen species (ROS) measurement
HT22 cells were seeded onto 96-well black walled microtiter plates at a density of 5 × 10 3 cells per well. The next day, the cells were treated with the 2.5-10 µM sterubin, eriodictyol or homoeridodictyol alone or in the presence of 5 mM glutamate for 7 h. The medium was then replaced with 100 µl loading medium (phenol red-free Hank's balanced salt solution containing 10 µM CM-H 2 DCFDA). After 30 min in CM-H 2 DCFDA (C6827, Invitrogen), the fluorescence (λ excitation = 495 nm, λ emission = 525 nM) was determined using a Molecular Devices microplate reader (San Jose, CA, USA). Each treatment was done in sextuplicate. DCFDA fluorescence was normalized to control cells not exposed to metals or glutamate.

Iron binding
Ferrous iron binding was measured using ferrozine as described [21]. Briefly, different dilutions of sterubin, eriodicyol and homoeridictyol were mixed with an equal volume of 5 µM FeCl 2 in 50 mM HEPES, pH 7.5 in a 96 well plate. After 2 min, 50 µl of 5 mM ferrozine was added and the absorbance at 562 nm measured on a Molecular Devices microplate reader. The amount of remaining iron was calculated from the difference in absorbance between the sample with ferrozine and the same sample without ferrozine divided by the difference between the control sample (iron plus ferrozine) and its blank (iron alone).

Western blots 2.7.1. Sample preparation
For Western blotting, 3 × 10 5 HT22 cells per 60 mm dish were grown for 24 h prior to the indicated treatments. For nuclear extracts, cells were rinsed twice in ice-cold Tris-buffered saline (TBS), scraped into ice-cold nuclear fractionation buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM Na 3 VO 4 , 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail) and incubated on ice for 15 min. Then NP40 at a final concentration of 0.6% was added, cells were vortexed and the nuclei pelleted by centrifugation. Nuclear proteins were extracted by sonication of the nuclear pellet in nuclear fractionation buffer and the extracts were cleared by additional centrifugation. Total protein extracts were prepared by rinsing the cells twice with ice-cold phosphate-buffered saline. The cells were scraped into lysis buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 50 mM NaF, 10 mM Na pyrophosphate, 5 mM EDTA, 1% Triton X-100, 1 mM Na 3 VO 4 , 1× protease inhibitor cocktail, 1× phosphatase inhibitor cocktail) and incubated on ice for 30 min. Extracts were sonicated and cleared by centrifugation. The supernatants were stored at − 70°C until analysis. Protein concentrations were quantified by the bicinchoninic acid method (Pierce) and adjusted to equal concentrations. 5× Western blot sample buffer (74 mM Tris-HCl, pH 8.0, 6.25% SDS, 10% β-mercaptoethanol, 20% glycerol) was added to a final concentration of 2.5× and samples were boiled for 5 min.

Western blotting
For SDS-PAGE, equal amounts of cellular protein, typically 10-20 µg per lane, were used. All samples were separated using 10% Criterion XT Precast Bis-Tris Gels (Biorad, Hercules, CA). Proteins were transferred to nitrocellulose membranes and the quality of protein measurement, electrophoresis and transfer checked by staining with Ponceau S. Membranes were blocked with 5% skim milk in TBS-T (20 mM Tris buffer pH 7.5, 0.5 M NaCl, 0.1% Tween 20) for 1 h at room temperature and incubated overnight at 4°C in the primary antibody diluted in 5% BSA in TBS/0.05% Tween 20. The primary antibodies used were: HRPconjugated rabbit anti-actin (#5125, 1/20,000) from Cell Signaling The results for the 9 most potent plant extracts in the primary screen (oxytosis assay using HT22 cells) as well as the BV-2 cell assay for anti-inflammatory activity and the PC12 cell differentiation assay. The effects of the extracts on the HT22 cell number was also determined. EC 50 = half maximal effective concentration. Subsequently, blots were washed in TBS/0.05% Tween 20 and incubated for 1 h at room temperature in horseradish peroxidase-goat anti-rabbit or goat anti-mouse (Biorad) diluted 1/5000 in 5% skim milk in TBS/0.1% Tween 20. After additional washing, protein bands were detected by chemiluminescence using the Super Signal West Pico Substrate (Pierce). For all antibodies, the same membrane was reprobed for actin or an antiserum reacting with the total protein.
Autoradiographs were scanned using a Biorad GS800 scanner. Band density was measured using the manufacturer's software. Relative protein expression was normalized to actin band density. Each Western blot was repeated at least three times with independent protein samples.

Determination of the Trolox equivalent activity concentration (TEAC)
TEAC values were determined as previously described [22].

Statistical analysis
Data from at least three independent experiments were normalized, pooled and analyzed using Graph Pad Prism 4 software followed by the statistical tests indicated in the figure legends. P < 0.05 was taken as significant.  The EC 50 s for the three flavonoids found in Yerba santa (sterubin, eriodictyol, homoeriodictyol) along with fisetin as a positive control in the 5 different phenotypic screening assays as well as their TEAC values, a measure of direct anti-oxidant activity, were determined as described in Section 2.

Phenotypic screening of plant extracts
We used a commercial plant extract library from Caithness Biotechnologies (http://caithnessbiotechnologies.com/) as the starting point for this study. This library was designed to maximize the potential for hit finding against diverse molecular targets while minimizing the content of potentially toxic compounds by focusing only on plants with a history of use as traditional medicines or a history of inverse association with disease risk. Moreover, this library has been carefully sized to optimally balance high molecular diversity and potential for lead finding by smaller research groups. Furthermore, the library contains only extracts from plants that are commercially available thereby negating any potential issues with recollection, sustainability or sovereignty of the discovery.
The library contains a total of 400 different plant extracts with two pre-fractions per sample, one polar and one non-polar. For this study, we focused on the non-polar extracts which were screened at a final dilution of 1/10,000 in the oxytosis assay using HT22 hippocampal nerve cells plated in 96 well plates [3]. Because of the generality of the toxicity pathway in oxytosis and its mechanistic association with both aging and AD, it is a good assay to quickly and simply screen the different extracts. Using this approach, we identified 9 extracts that provided significant protection from glutamate at the dilution used (Table 1). Further analysis of these extracts for neurotrophic activity (PC12 differentiation) and anti-inflammatory activity (BV2 cells) as well as direct toxicity (Table 1) identified two extracts, C and G, that not only were highly protective in the oxytosis assay but also had strong anti-inflammatory and neurotrophic activity and showed < 10% cell growth inhibition at the protective doses. Here we report our studies on C whose source, Yerba santa (Eriodictyon californicum), is a shrub native to California and thus of significant local interest.

HPLC fractionation and screening in the oxytosis assay
Given the complex composition of compounds in a crude extract, C was fractionated by HPLC (Fig. 1A) and each fraction was then prepared identically and tested at the same dilution in the oxytosis assay (Fig. 1B). Fraction 17 was identified as the one that protected the most with~50% cell survival (Fig. 1B). No other fractions showed any significant protection. Therefore, this fraction was selected for further study.

Identification and purification of the flavonoid sterubin
In order to determine the composition of fraction 17 and possibly identify individual biologically active compounds, the fraction was analyzed by mass spectrometry. A strong peak was identified with a molecular weight (MW) identical to two flavonoids known to be present in Yerba santa, homoeriodictyol and sterubin (MW = 302) (Fig. 1C) [23]. The mass fragmentation pattern was also determined to serve as a fingerprint to identify the compound in the active fraction (Fig. 1D). Homoeriodictyol and sterubin were obtained from commercial sources and their HPLC elution times and mass fragmentation patterns determined. While both characteristics of homoeriodictyol were distinct from those of the active fraction, those of commercially sourced sterubin (Fig. 1E) were almost identical strongly suggesting that the active compound of this fraction was sterubin.

Validation of the biological activity of sterubin in screening assays
In order to determine whether the potent biological activity of C could be explained by the presence of sterubin, the biological activity of the purified compound was tested in all of the different screening assays ( Table 2). For comparison, the EC 50 values for homoeriodictyol and eriodictyol, two other flavonoids which are also present in Yerba santa and have been much more widely studied, are also shown. In addition, the EC 50 values for fisetin are included. Fisetin is a neuroprotective and cognition-enhancing molecule which our laboratory has identified using some of the same screening assays [14,24] and that we have recently shown to protect in in vivo models of dementia and AD [25,26]. The results show that sterubin is indeed a highly effective compound in all of the assays. In particular, sterubin is very effective at inhibiting intracellular Abeta toxicity with an EC 50 less than 1 µM. While eriodictyol has weaker but still significant activity in the same assays, homoeriodictyol is completely inactive at concentrations up to 10 µM. Importantly, the EC 50 values of sterubin were substantially better than those of fisetin.

Mechanisms underlying the beneficial activities of sterubin
The steps involved in oxytosis have been well characterized. The first step involves the loss of the major endogenous intracellular antioxidant glutathione (GSH). Thus, we first asked if sterubin could preserve GSH levels. As shown in Fig. 2, sterubin dose-dependently increased GSH levels in control cells ( Fig. 2A, B) and maintained the levels of GSH in cells treated with glutamate to induce oxytosis ( Fig. 2A,  B). Eriodictyol was less effective at both increasing control GSH levels and maintaining GSH in the presence of glutamate (Fig. 2B) while homoeriodictyol was completely ineffective (Fig. 2B). Consistent with the ability of sterubin to maintain GSH levels, it also provided excellent, dose-dependent protection against two inducers of ferroptosis, a cell death pathway closely related, if not identical, to oxytosis [7]. Erastin, similar to glutamate, blocks system xc-activity while RSL3 is an inhibitor of glutathione peroxidase 4 that is downstream of GSH loss and plays a key role in the cell death process in both oxytosis and ferroptosis [7] (Fig. 2C).
Downstream of GSH loss in oxytosis is an increase in ROS levels. As shown in Fig. 2D, sterubin strongly and dose-dependently reduced glutamate-induced ROS production. Eriodictyol was less effective (Fig. 2D) and homoeriodictyol had no effect (not shown). Interestingly, sterubin also significantly reduced the basal levels of ROS (Fig. 2D) prompting an examination of the effects of sterubin against two direct pro-oxidants, H 2 O 2 and t-butyl peroxide (tBOOH), both of which can also promote cell death. As shown in Fig. 2E, sterubin was surprisingly effective against cell death induced by either H 2 O 2 or tBOOH with EC50s of~1 µM. These results prompted the measurement of the sterubin's TEAC value which was quite low (Table 2) suggesting that it is unlikely to act as a direct antioxidant.
Iron can promote cell death through its ability to generate ROS. Since ferroptosis is described as an iron dependent form of cell death [27] and iron chelators also protect from oxytosis [7], we next asked if sterubin can chelate iron, which might contribute to its antioxidant actions. To test its iron binding, we used the ferrozine assay [21] which looks at the ability of a compound to prevent iron binding to ferrozine. As shown in Fig. 2F, sterubin is quite an effective iron chelator even at ratios of 1:1 flavonoid:iron. While eriodictyol showed similar activity, homoeriodictyol demonstrated very little iron binding even at a ratio of 10:1 flavonoid:iron.
Two transcription factors contribute to the upregulation of GSH, Nrf2 and ATF4 [5]. Thus, it was next asked if sterubin can alter the levels of these proteins in the HT22 cells. As shown in Fig. 3A, sterubin dose-dependently increased nuclear Nrf2 levels to a maximum of 6-fold at 5 µM. This increase was seen within 30 min of sterubin treatment and remained constant for up to 6 h (not shown). Eriodictyol also induced Nrf2 with similar efficacy as sterubin (Fig. 3B). In contrast, homoeriodictyol had a much lesser effect on Nrf2 levels (not shown). Since we have shown that ATF4 plays an important role in the maintenance of GSH levels in nerve cells [28], we also examined the effects of the three flavonoids on ATF4 levels using the same nuclear extracts. As shown in Fig. 3C, sterubin dose-dependently increased nuclear ATF4 levels to a maximum of 3-fold at 5 µM and eriodictyol had a similar effect. As with Nrf2, the increase in nuclear ATF4 was seen within 30 min of treatment Nrf2 regulates the expression of multiple enzymes involved in GSH metabolism and protection against oxidative stress [for review see [29]]. Thus, we examined the levels of some of these proteins by Western blotting. As observed in Fig. 3D, 10 µM sterubin increased the levels of one of the glutamate cysteine ligase (GCL) subunits (GCLM) within 6 h of treatment (1.37-fold). In addition to GCLM, sterubin increased the levels of p62 (22.7-fold) and heme oxygenase 1 (HO-1) (4.1fold), two proteins involved in protection against oxidative stress and known to be regulated by Nrf2 [30,31] with a similar time course (Fig. 3D) further demonstrating that sterubin induces a variety of antioxidant proteins.
Yerba santa has long been known for its anti-inflammatory properties [32] and in our phenotypic screens we found that sterubin had potent anti-inflammatory activity in BV-2 microglial cells whereas eriodictyol was less effective and homoeridodicytol had no effect at concentrations up to 10 µM (Table 2). To expand upon this observation, the BV-2 cells were treated with different doses of sterubin or eriodictyol in the presence of LPS and the production of NO, IL-6, IL-1β and TNFα were determined on culture supernatants and the levels of iNOS and COX2 assessed in cell extracts. Sterubin strongly and dose-dependently reduced the production of NO, IL-6 and IL1β (Fig. 4A-C) as well as the levels of both iNOS and COX2 (Fig. 5A, B). A lesser effect was seen on the induction of TNFα (Fig. 4D). Eriodictyol was much less effective ( Fig. 4A-D) and homoeriodictyol, even at 5-10 µM, had little or no effect on any of these parameters (not shown). Sterubin also dosedependently increased nuclear Nrf2 and ATF4 levels in the BV-2 cells ( Fig. 5C-E) suggesting that either or both of these transcription factors might be a common target for both its neuroprotective and anti-inflammatory effects.
To test this idea, we first downregulated Nrf2 in the HT22 nerve cells and the BV-2 microglia using specific siRNA. In the HT22 cells, the Nrf2 levels were decreased by~90% (Fig. 6A) while in the BV-2 cells the Nrf2 levels were decreased by~80% (Fig. 7A). In the HT22 nerve cells, knockdown of Nrf2 significantly shifted the dose-response curve for neuroprotection by sterubin in the oxytosis assay (Fig. 6B). In contrast, Nrf2 knockdown had little or no effect on the protective effects of sterubin against H 2 O 2 or tBOOH (Fig. 6B, C) suggesting that these actions were mediated by as yet unidentified pathways. In the BV-2 cells, the effects of Nrf2 knockdown were much more dramatic with the EC 50 s for inhibition of NO, IL-6 and IL-1β production shifted from 1-2 µM to greater than 10 µM (Fig. 7B-D). Thus, Nrf2 appears to play a key role in the anti-inflammatory effects of sterubin and contribute to its neuroprotective activity in the HT22 cells.
Since sterubin also induces ATF4 (Fig. 3), we looked at the effects of ATF4 knockdown on the activities of sterubin in both BV2 microglia and HT22 nerve cells. In contrast to the results with Nrf2 knockdown, loss of ATF4 by 80-85% in the HT22 cells (Fig. 6A) had little or no effect on neuroprotection against glutamate (Fig. 6) and actually enhanced the neuroprotective effects of sterubin against H 2 O 2 or tBOOH (Fig. 6B, C). These results further support the idea that these actions by sterubin are mediated by pathways distinct from those involving ATF4 or Nrf2. Similarly, loss of ATF4 by~80% (Fig. 7A) had no effect on the anti-inflammatory activity of sterubin (Fig. 7B-D).

Discussion
In this manuscript, we show for the first time that the most significant neuroprotective flavonoid in Yerba santa is the flavanone sterubin. Although sterubin has long been known to be present in Yerba santa [23], it has remained remarkably unstudied with most reports focusing on the other Yerba santa flavonoids, eriodictyol or homoeriodicytol. Moreover, studies which have identified sterubin in various screens have often ignored it based on the incorrect assumption that it was already well studied [eg 33]. While eriodictyol has some of the same properties as sterubin, albeit with significantly higher EC 50 s, homoeriodictyol is essentially inactive in most of our assays at concentrations up to 10 µM. Yerba santa has long been known to have medicinal properties. For example, a treatise published in 1902 on plants used by the native people in Mendocino County, California [34] states that "No plant is more highly valued as a medicine by all the tribes of Mendocino County." Historically, it was used to treat a variety of indications including various respiratory conditions and fever as well as bruises, infections and pain including headaches [32,34]. Thus, its anti-inflammatory properties are well documented, consistent with the potent anti-inflammatory effects of sterubin in our in vitro assay.
Although sterubin has not previously been studied in the context of neuroprotection, there are a number of reports on eriodictyol. However, consistent with our results, eriodictyol was previously shown not to be effective either against oxytosis in HT22 cells or LPS-induced nitrite production in BV-2 microglial cells [35]. Nevertheless, other studies have found that eriodictyol could reduce both oxidative stress and inflammation in both cell culture and animal models [36][37][38][39]. The in vitro neuroprotective effects were at least partially dependent on Nrf2 [36,37] but required relatively high concentrations of the flavonoid (> 20 µM). This is in contrast to the strong effects seen in this study with sterubin even at 1-2 µM. Although it has recently been reported that homoeriodictyol can also induce Nrf2 and protect against oxidative stress in endothelial cells [40], we did not observe a similar effect in the HT22 or BV-2 cells.
Previously, we showed that the flavonol fisetin could upregulate both Nrf2 and ATF4 [41] and, at the time, this appeared to be the first molecule described which could do so. As shown in this manuscript, sterubin, as well as eriodictyol, exhibit a similar property. However, in contrast to fisetin [41], Nrf2 rather than ATF4 plays a role in the neuroprotective effects of sterubin. Nrf2 is also critical to the anti-inflammatory actions of sterubin. Nrf2 has long been known to have both cytoprotective [31] and anti-inflammatory activity [42]. The cytoprotective activity is related to its ability to simultaneously upregulate the transcription of genes encoding a variety of antioxidant and detoxification proteins including those that regulate intracellular GSH homeostasis, major components of the thioredoxin system which modulates protein oxidation, and proteins involved in heme and iron metabolism. A number of these proteins, such as HO-1, also contribute to the anti-inflammatory effects of Nrf2 [42]. Nrf2 can also directly suppress the transcription of some pro-inflammatory cytokines including IL-6 and IL-1β [43] consistent with our data in the BV-2 cells.
In addition, we show that sterubin is an effective iron chelator. Since iron can contribute to nerve cell damage in aging and neurodegenerative diseases [8], this property is likely to contribute to the neuroprotective activity of sterubin both in vitro and potentially in vivo. Importantly, we recently showed that iron can potentiate GSH loss and cell death [20] in response to compounds that promote GSH loss but not in response to other oxidants. This finding might help to explain at least some of the protective activity of sterubin that is not dependent on Nrf2 in the glutamate-treated HT22 cells.
We also show that sterubin is effective against multiple inducers of cell death that activate distinct cell death pathways. Importantly, not only is sterubin very effective against oxytosis induced by glutamate treatment but also against inducers of the very closely related ferroptotic pathway [7] including erastin which has the same target as glutamate and RSL3 which inhibits glutathione peroxidase 4, a downstream target of this pathway [7]. This finding indicates that not only does sterubin protect against oxytosis/ferroptosis by maintaining GSH levels under conditions of stress but it also acts downstream of GSH loss. In addition, sterubin prevents cell death induced by both H 2 O 2 and tBOOH, although in contrast to the protection against oxytosis, Nrf2 does not seem to be involved in sterubin-mediated protection against these two agents. This protection may involve the iron chelating activity of sterubin as iron chelators can protect against H 2 O 2 and tBOOH toxicity (not shown). Importantly, sterubin is also very effective at protecting the MC65 nerve cells against the toxic accumulation of intracellular amyloid beta peptide which is considered by many to be a primary toxic event in AD [17].
An important property that distinguishes sterubin from either eriodictyol or homoeriodictyol is its ability to induce PC12 cell differentiation (Table 2). We [3] and others [44] have suggested that the ability to activate neurotrophic signaling pathways is an important characteristic of potential neuroprotective compounds. Indeed, treatments that only activate anti-oxidant systems may not be adequate to address the numerous changes that occur in the aging brain and that are exacerbated in AD. Thus, a compound such as sterubin that not only activates global anti-oxidant systems via Nrf2 but also mimics neurotrophic signaling may be particularly effective for the treatment of neurodegenerative diseases.
By combining the analyses of the neuroprotective activities of sterubin, eriodictyol, homoeriodictyol, and fisetin in our phenotypic screening assays (Table 2) with their physicochemical properties (Table 3), we were able to elaborate a plausible structure-activity relationship (SAR). The catechol B-ring with phenolic hydroxyls on sterubin is essential for its neuroprotective activities. Comparing their chemical structures, sterubin, eriodictyol, and fisetin contain a 3′,4′dihydroxy on the catechol B-ring and they are all active in our cellbased assays with EC 50 values less than 5 µM. In contrast, homoeriodictyol, which contains a 3′-methoxy 4′-hydroxy on the B-ring, is inactive (EC 50 s > 10 µM). For flavonoids, free phenolic hydroxyl groups on the catechol ring provide hydrogen bonds important for molecular recognition. They are key structural features known to be responsible for the radical scavenging, anti-oxidant, anti-inflammatory and anti-protein aggregation activities [45,46]. Because our phenotypic screens in cell culture models mimic several aspects of the changes seen in the aging brain including increases in oxidative stress, energy loss, neuroinflammation, and intracellular Aβ aggregation, it is logical that we identified sterubin, eriodictyol, and fisetin as being active in these assays. Interestingly, sterubin has a relatively low TEAC value supporting the idea that it protects from oxidative stress-induced death via an indirect anti-oxidant mechanism. In addition, the ortho-dihydroxyls on the B-ring play a role in chelation of oxidizing metal ions [45]. This was confirmed in the present study which showed that sterubin and eriodictyol effectively bind Fe 2+ in a dose-dependent manner while homoeriodictyol does not (Fig. 2F). On the other hand, the hydroxyl groups in the A-ring of sterubin are not critical for the neuroprotection but could play a modulating role. Sterubin is a derivative of eriodictyol where the only difference is the 7-methoxy group on the A-ring of sterubin. Such O-methylation on the A-ring increases the overall lipophilicity of sterubin (ClogP, 2.37) compared to eriodictyol (ClogP, 1.85). It also decreases the total polar surface area (tPSA) from 107 to 96 Å and reduces the number of hydrogen bond donors (HBD) to 3 ( Table 3). It is conceivable that such changes in its physiochemical properties increase, at least in part, the passive membrane permeability of sterubin for better target engagement in cells, which in turn contributes to the approximately 3-fold increase of neuroprotective potency in comparison with eriodictyol ( Table 2). In addition, methylation of flavonoids has been reported to improve their bioavailability and metabolic stability [47].
Sterubin is also about 4-fold more potent in the phenotypic screening assays than fisetin, an AD drug candidate [25,26]. Despite their structural differences in the A-and C-rings, sterubin has a higher ClogP, a smaller tPSA, and one less HBD compared to fisetin. Again, in support of our hypothesis, this may result in sterubin being more cell membrane permeable and thereby more potent than fisetin. Touil et al. [48] previously reported that geraldol, a 3′-O-methylated derivative of fisetin, is an active metabolite in mice and shows a higher potency of cytotoxic and antiangiogenic activities in vitro. Although those compounds were tested for anticancer rather than neuroprotective activities, this provides additional evidence that methylation of flavonoids can modulate their physiochemical properties and potency and the methylation position may play a distinct roles in modulating different biochemical. Strikingly, sterubin meets the desirable physicochemical criteria to be a viable CNS drug candidate (Table 3), and it has a higher score of CNS druglikeness than fisetin [49]. However, it will be important to investigate the sterubin metabolites and pharmacokinetics in vivo in order to provide a better understanding of the pharmacological behavior of sterubin.
Finally, one of the known properties of sterubin that overlaps with those of eriodictyol and homoeriodictyol is its bitter masking property [23]. Studies have shown that all three flavonoids are antagonists of bitter taste receptors (T2Rs) [50]. While bitter receptors are best known and studied for their role in taste, recent studies have shown that these receptors are also located in multiple regions of the brain [51] where their roles are presently unknown. However, there is evidence for the upregulation of some of these receptors in AD as well as other neurodegenerative diseases [52]. Thus, a compound such as sterubin which can act as a T2R receptor antagonist might have additional, and as yet Table 3 Structures and physiochemical properties of sterubin, eriodictyol, homoeriodictyol and fisetin.

undescribed, beneficial effects in vivo.
Together these studies provide further support for the use of our set of phenotypic screening assays to identify more potent neuroprotective molecules. In this report, we used a curated library of plant extracts and found that 9/400 (2.25%) extracts were highly active. Using the oxytosis assay as our primary screen, we identified extracts that are extremely effective against a form of cell death that is associated with a variety of age-associated diseases including AD [7]. The subsequent identification of sterubin in one of the most active extracts shows that even compounds which are generally thought of as well studied [33] can provide surprising results. Thus, based on its potent Nrf2-inducing as well as neurotrophic, anti-inflammatory and iron chelating properties, we believe that sterubin deserves further examination in the context of AD, as well as other neurodegenerative diseases.


Protein scaffolded DNA tetrads enable efficient delivery and ultrasensitive imaging of miRNA through crosslinking hybridization chain reaction† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc01001c

DNA tetrads as an integrated platform that enables realization of efficient DNA delivery and HCR amplification in situ on nanostructures.

Electrophoresis was performed at a constant voltage of 100 V for 2 h with a load of 10 µL of sample in each lane. After electrophoresis, the gel was visualized using a Tocan 240 gel imaging system (Tocan Biotechnol. Co., Shanghai, China).

In vitro fluorescence assay
To prepare DNA hairpins for DNA tetrads, the all hairpins were kept at 95 °C for 5 min, then quickly treated with ice for 5 min, and finally transferred to room temperature for 2 h before further use. 1x PBS solution was used to dissolve the hairpins. Subsequently, appropriate SA were added to form DNA tetrads. DNA tetrads was incubated with RNA target of a varied concentration in 50 L 1×TAE-Mg 2+ buffer at 37 °C for 4 h. Fluorescence spectra were recorded using a Fluorescence spectrometer (Jobin Yvon Horiba) with excitation at 540 nm. The slit was set to be 5 nm for both the excitation and the emission.
To evaluate its selectivity, the DNA tetrads was also used for detecting other co-existing cellular components including miR-210, miR-214, let-7a, 100 nM β-actin protein, L-02 cells lysate. Additionally, one more sample was incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum.

Cell culture and confocal microscopy imaging
HeLa cells and L-02 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U mL -1 penicillin, and 100 U mL -1 streptomycin. HepG2 cells were incubated in DMEM supplemented with 10% fetal bovine serum.
All cell lines were maintained at 37 °C in a 100% humidified atmosphere containing 5% CO 2 . The cell density was determined using a TC20 automated cell counter (BIO-RAD, USA).
Fluorescence imaging of cells was performed as follows: The cells were washed three times with 1x PBS, then incubated with a fresh medium containing the given amount of DNA tetrads or Lipofectamine 3000 at 37 °C for 4 h. After washing three times with 1x PBS, the cells were incubated with 1 mL fresh medium at 37 °C before imaging.
Fluorescence imaging of lysosome escape of DNA tetrads was performed as follows: HeLa cells were incubated with 500 L culture medium containing the given amount of DNA tetrads at 37 °C for varying time. After washing three times with 1x PBS, the cells were incubated with fresh RPMI 1640 medium containing 50 nM Lysosome traker (Lyso@tracker Red) for 10 min followed by imaging.
Fluorescence imaging of cellular uptake for DNA tetrads was performed as follows: Cells were incubated with 500 L culture medium containing the given amount of DNA tetrads at 37 °C for 1 h. The cells were washed three times with 1x PBS before imaging. For cellular uptake experiment at 4 °C, cells were first incubated at 4 °C for 0.5 h and then grown in 500 L culture medium containing the given amount of DNA tetrads at 4 °C for 1 h followed by imaging. For cellular uptake experiment in the presence of NaN 3 , cells were first pretreated at 37 °C for 1 h with 1 mL culture medium containing 1% NaN 3 and then incubated with 500 L culture medium containing the given amount of DNA tetrads and 1% NaN 3 at 37 °C for 1 h followed by imaging.
All fluorescence images were acquired using an oil dipping objective on Nikon TI-E+A1 SI confocal laser scanning microscope (Japan). Fluorescence of Cy3 and LysoTracker Red DND-99 was obtained by excitation at 561 nm and collected over 570-620 nm; Fluorescence of Cy5 was obtained by excitation at 640 nm and measured over 670-720 nm; Fluorescence of FRET was obtained by excitation at 561 nm and measured over 670-720 nm.

qRT-PCR quantification of miR-21 expression
Total cellular RNA was extracted from HepG2, HeLa, L-02 cells using the SanPrep Column microRNA Mini-Preps Kit  Hairpin probe H1 Biotin-TTT TTT TTT TTT TTT TTC Figure S1. 3% Agarose gel to compare the purity of the DNA nanotetrads before and after agarose gel purification.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Chemistry

text:
Suppressive effect of syndecan ectodomains and N-desulfated heparins on osteoclastogenesis via direct binding to macrophage-colony stimulating factor

Syndecans, a family of cell surface heparan sulfate proteoglycans, regulate cell differentiation via binding of their heparan sulfate chains to growth factors and cytokines and play a role in tumor growth and progression, wound repair, and intestinal mucosal damage. However, the functional and mechanistic roles of syndecans in osteoclast differentiation and bone metabolism are yet unclear. Here, we demonstrated that post-translationally glycosylated ectodomains of syndecan-1 to 4 obtained from mammalian cells efficiently suppressed osteoclast differentiation compared to those obtained from Escherichia coli with no systems for glycosylation. A concomitant decrease in the expression of osteoclast markers such as nuclear factor of activated T cells 1 (NFATc1), c-Fos, and ATP6V0D2 was observed. In addition, heparan sulfate and selectively N-desulfated heparin derivatives with 2-O- and 6-O-sulfate groups and no anticoagulant activity in blood inhibited osteoclast differentiation. The inhibitory effects of syndecan ectodomains, heparan sulfate, and N-desulfated heparin derivatives on osteoclast differentiation were attributed to their direct binding to the macrophage-colony stimulating factor (M-CSF), resulting in the blocking of M-CSF-mediated downstream signals such as extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), p38, and Akt. Furthermore, mice injected with syndecan ectodomains, heparan sulfate, and N-desulfated heparin derivatives into periosteal regions of calvaria showed reduction in the formation of tartrate-resistant acid phosphatase (TRAP)-positive mature osteoclasts on the calvarial bone surface, thereby exhibiting decreased bone resorption. Together, these results revealed a novel role of heparan sulfate chains of syndecan ectodomains in the regulation of osteoclast differentiation.

Introduction
Bone formation and remodeling are regulated by the complex interplay between osteoblasts and osteoclasts in the bone marrow environment. The excessive bone resorption by osteoclasts relative to bone formation by osteoblasts leads to pathologic bone loss such as in osteoporosis, periodontal disease, cancer-associated bone disease, rheumatoid arthritis, and Paget's disease 1 . Osteoclastogenesis is primarily regulated by two cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear κB ligand (RANKL), which are expressed and released by osteoblasts in the bone marrow 2,3 . M-CSF functions in the proliferation and differentiation of osteoclast precursors by binding to its receptor c-Fms localized on the cytoplasmic membrane of osteoclast precursors and subsequently stimulating diverse signal transduction pathways, including Akt and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-JUN Nterminal kinase (JNK), and p38 4 . RANKL promotes mononuclear osteoclast precursor fusion, followed by multinucleated osteoclast maturation 3,5 .
Proteoglycans comprise a protein core that may covalently attach to at least one glycosaminoglycan (GAG) chain at specific sites 6 . GAGs are unbranched polysaccharides with a backbone of repeating disaccharide units and classified into four groups, namely, heparin/ heparan sulfate, chondroitin sulfate/dermatan sulfate, keratan sulfate, and hyaluronic acid 7 . Among various constituents of proteoglycans, heparan sulfate proteoglycans, which play an essential role in embryological development and normal physiology, have enormous structural diversities in heparan sulfate chains in terms of chain length and size, saccharide composition and arrangement, and sulfation and epimerization pattern within the sugar segments 8,9 . Distinct structures of heparan sulfate chains in the same core protein are produced in different cell types and at different tissue sites 10 . Heparan sulfate chains have been found to bind to a wide range of cellular components such as fibroblast growth factors (FGFs) and their receptors, transforming growth factors, interleukins, bone morphogenetic proteins, lipases and apolipoproteins, and extracellular matrix proteins 9,11 .
Syndecans, a family of heparan sulfate proteoglycans that are represented by four members having similar structural organization (syndecan-1 to 4) in mammals, have been reported to regulate cell adhesion, spreading, migration, proliferation, survival, and differentiation as well as the maintenance of cell morphology 12,13 . Syndecan-1 and syndecan-3 are mainly expressed in the epithelial and neuronal cells, respectively 12,14 . Syndecan-2 is mostly present in mesenchymal and smooth muscle cells and syndecan-4 is ubiquitously expressed 15 . Syndecans comprise three distinct domains, including a C-terminal cytoplasmic domain, transmembrane domain, and N-terminal extracellular domain (ectodomain). The ectodomain of syndecans contains heparan sulfate and chondroitin sulfate GAG side chains and exhibits the ability to bind with growth factors and cytokines such as FGF-2, hepatocyte growth factor (HGF), and interleukin-8 [16][17][18][19] . All syndecans are shown to carry heparan sulfate chains, and syndecan-1 and syndecan-3 display both heparan sulfate and chondroitin sulfate chains in the ectodomain 20 . Extracellular shedding of syndecans to generate soluble ectodomains has been reported to constitutively occur under physiological conditions or may be significantly induced by stimuli-mediated activation of various proteases such as matrix metalloproteinase-2 (MMP-2), MMP-7, MMP-9, and membrane-associated MT1-MMP and MT3-MMP [21][22][23] . Shed ectodomains of syndecans were shown to play an important role in pathophysiological events such as tumor growth and progression, wound repair, and intestinal mucosal damage 19,24,25 . Syndecan-1 ectodomains from myeloma cell lines were shown to inhibit osteoclast formation and induce osteoblast development in bone marrow cell cultures 26 , although the detail mechanisms remain largely unknown.
In this study, we identified that syndecan ectodomains and heparan sulfate chains negatively regulate osteoclast differentiation via direct interaction with M-CSF, as evident from a pull-down, an immunosorbent-based, and a competitive binding assay. In addition, selectively N-desulfated heparin derivatives with no anticoagulant activity suppressed osteoclastogenesis. Consistent with the results of in vitro studies, the subcutaneous administration of syndecan ectodomains, heparan sulfate, and N-desulfated heparin derivatives into mouse calvaria resulted in a decrease in osteoclast formation and bone resorption on the surface of the calvarial bone. From these results, we elucidated the functional mechanism underlying the inhibitory action of heparan sulfate of syndecan ectodomains on osteoclast differentiation.

Syndecan ectodomains suppress osteoclast differentiation
The ectodomains of mouse syndecan-1 to 4 (mSDC1ED to mSDC4ED) were expressed in two different cell types, mammalian HEK293E cells and E. coli Rosetta 2 (DE3) strain. The corresponding amino acids of syndecan ectodomains from HEK293E cells and E. coli are presented in Fig. 1a and Supplementary Fig. 1a (left panel), respectively. The molecular weights of ectodomains of syndecan-1, 2, 3, and 4 from HEK293E cells were approximately 70, 37.5, 120, and 40 kDa, respectively, and those of ectodomains of syndecan-1, 2, 3, and 4 from E. coli were approximately 42, 25, 70, and 30 kDa, respectively ( Fig. 1a and Supplementary Fig. 1a; right panel). Despite the similarity of predicted molecular weights of syndecans expressed in two different cell expression systems, the molecular weights of syndecan ectodomains from HEK293E cells were much higher than those of ectodomains from E. coli, suggesting that syndecan ectodomains expressed in mammalian cells may carry heparan sulfate and chondroitin sulfate side chains. These observations are consistent with previous reports, wherein syndecanbearing heparan sulfate and chondroitin sulfate chains exhibited higher molecular weights than their expected sizes 26,27 .
We next examined the effect of syndecan ectodomains on the differentiation of bone marrow-derived osteoclast precursors into osteoclasts in the presence of M-CSF and RANKL. Recombinant ectodomains of syndecan-1 to 4 from HEK293E cells and E. coli inhibited osteoclast differentiation in a dose-dependent manner ( Fig. 1b and Supplementary Fig. 1b), with a simultaneous decrease in the expression level of osteoclastogenic marker genes such as nuclear factor of activated T cells 1 (NFATc1), c-Fos, and ATP6V0D2 (Fig. 1c). The maximum inhibitory dose of syndecan ectodomains from HEK293E cells was 1 nM, whereas bacterially produced syndecan ectodomains lacking GAG side chains failed to block osteoclast differentiation at the same concentration. This observation suggests that heparan/chondroitin sulfate chains of syndecan ectodomains may play a critical role in osteoclastogenesis. On the contrary, syndecan ectodomains from E. coli suppressed osteoclast differentiation at concentrations ranging from 100 to 6000 nM (Supplementary Fig. 1b), indicating that much higher concentrations of core proteins of syndecan ectodomains may contribute to the regulation of osteoclast differentiation in a different way from GAG side chains. To determine the time point of the inhibitory action of syndecan ectodomains during the multi-step process of osteoclast differentiation, cells were treated with syndecans at different time points post-differentiation. The inhibitory effect was effective when syndecans were treated at the early stage (day 0 to 1) of osteoclast differentiation, whereas such a phenomenon disappeared with the progression of differentiation (Fig. 1d). In addition, we observed that syndecans blocked the activity of tartrateresistant acid phosphatase (TRAP), an early marker of osteoclastogenesis, in a dose-dependent manner (Supplementary Fig. 2). Taken together, syndecan ectodomains produced in mammalian cells may suppress the early stage of osteoclastogenesis.

Syndecan ectodomains inhibit osteoclastogenesis through direct interaction with M-CSF
Syndecans have been shown to be involved in the regulation of cell proliferation 16,24,26,27 . To analyze Osteoclast precursors were treated with syndecan ectodomains (1 nM) in the presence of M-CSF and RANKL for 4 days. Cell lysates were subjected to immunoblot analysis using antibodies specific for NFATc1, c-Fos, and ATP6V0D2. β-Actin was used as a loading control. Asterisk indicates non-specific bands. d Osteoclast precursors were treated with syndecan ectodomains (1 nM) at the indicated time points during osteoclast differentiation. After TRAP staining, the number of TRAP(+) MNCs was counted. Results represent the means ± SD (n = 3). **p < 0.01 the inhibitory mechanisms of syndecan ectodomains on osteoclast differentiation, we first examined osteoclast precursor proliferation using MTT assay. Syndecan-1 to 4 ectodomains suppressed osteoclast precursor proliferation in the presence of M-CSF (Fig. 2a, upper  panel). However, syndecan ectodomains had no effect on the growth of RAW264.7 cells, which are able to proliferate in the absence of M-CSF (Fig. 2a, lower panel), or on their differentiation in the presence of RANKL alone ( Supplementary Fig. 3). In addition, syndecan-1 to 4 ectodomains strongly inhibited M-CSF-induced MAPKs (ERK, JNK, and p38) and Akt activation in osteoclast precursors (Fig. 2b), but did not affect RANKL-stimulated MAPKs activation (Supplementary Fig. 4). These findings suggest that the inhibitory effect of syndecan ectodomains on osteoclast differentiation ( Fig. 1) may be related to the defect in M-CSF signaling. Osteoclast precursors were pre-incubated with syndecan ectodomains (10 nM) for 4 h and stimulated with M-CSF (5 ng/mL). Whole cell lysates were immunoblotted with specific antibodies against p-ERK, ERK, p-JNK, JNK, p-p38, p38, p-Akt, and Akt. c Pull-down assay. His-tagged syndecan ectodomains from HEK293E cells and E. coli immobilized on Ni-NTA agarose beads were incubated with recombinant M-CSF and centrifuged. Bound proteins were separated by 15% SDS-PAGE and analyzed with immunoblot analysis with anti-M-CSF antibody (upper panel). Syndecan ectodomains retained in the resultant pellets were resolved by 10% SDS-PAGE and detected by staining with Coomassie Blue (lower panel). d Immunosorbent-based assay. His-tagged syndecan ectodomains from HEK293E cells were attached to the Ni-coated plate and incubated with M-CSF. After washing, the binding affinity of M-CSF to syndecan ectodomains was quantitatively analyzed by sequential incubations with anti-M-CSF antibody, HRP-labeled secondary antibody, and substrate. Absorbance was measured at 450 nm wavelength. e Crosslinking between syndecan ectodomains and extracellular matrix proteins. His-tagged syndecan ectodomains from HEK293E cells were added to vitronectin (VN) and osteopontin (OPN)-coated plates and further incubated for 1 h. After washing, the bound proteins were quantitatively measured as described in (d) using anti-His antibody. Results represent the means ± SD (n = 4 as in a; n = 3 as in d and e). The p value indicates the comparison between the treatment group and control. *p < 0.05; **p < 0.01 The heparan sulfate chains of syndecan ectodomains are known to bind to growth factors, including HGF and FGF-2 16,17 . Thus, we investigated whether syndecan-1 to 4 ectodomains could interact with M-CSF using two independent binding assays. In the pull-down assay, the mixture of M-CSF and His-tagged syndecan ectodomains was bound to Ni-NTA agarose beads. M-CSF directly bound to syndecan ectodomains from HEK293E cells but not to those produced from bacteria (Fig. 2c). In another binding analysis, His-tagged syndecan ectodomains immobilized on a Ni-coated microtiter plate were reacted with M-CSF. The interaction of M-CSF with syndecan ectodomains from HEK293E cells was much stronger than its interaction with syndecan ectodomains from E. coli (Fig. 2d). These results indicate that syndecan ectodomains expressed in mammalian cells seem to directly bind to M-CSF via their heparan/chondroitin sulfate side chains. As sulfated GAG chains, including heparan/ chondroitin sulfate, were reported to interact with extracellular matrix proteins 28-30 , we next examined whether syndecan ectodomains from HEK293E cells bind to matrix proteins such as vitronectin (VN) and osteopontin (OPN). Syndecan ectodomains effectively attached to VN and OPN upon their exposure to plates coated with VN or OPN (Fig. 2e). These results support the hypothesis that syndecan ectodomains bind to matrix proteins present in the bone marrow and that their complexes are able to recruit M-CSF to induce spatial sequestration of M-CSF in the extracellular compartment.
To assess the effect of syndecan ectodomains on osteoclastogenesis in animal models, we introduced syndecan ectodomains from HEK293E cells into the periosteum of mouse calvaria. Consistent with in vitro data, syndecan-1 to 4 ectodomains decreased the formation of TRAP-positive osteoclasts on the surface of the calvarial bone (Fig. 3a). Furthermore, the area of calvarial bone marrow cavity, an index of bone resorption, was decreased following administration of syndecan ectodomains (Fig. 3b). These findings indicate that syndecan ectodomains inhibit osteoclast formation, thereby attenuating osteoclastic bone resorption in vivo.

Heparan sulfate suppresses osteoclast differentiation via interaction with M-CSF
Previous reports have shown that syndecan ectodomains contain heparan sulfate and chondroitin sulfate chains that regulate cell growth and differentiation 16,26 . We first analyzed osteoclast proliferation in two different cell systems, bone marrow-derived osteoclast precursors and RAW264.7 cells growing in the presence and absence of M-CSF, respectively. Heparan sulfate effectively inhibited osteoclast precursor proliferation as compared with chondroitin sulfate (Fig. 4a, left panel). However, both heparan sulfate and chondroitin sulfate had no effect on the growth of RAW264.7 cells (Fig. 4a, right panel). Heparan sulfate strongly diminished the differentiation of osteoclast precursors into osteoclasts in a dose-dependent manner, but chondroitin sulfate had no remarkable effect on the inhibition of osteoclast differentiation (Fig. 4b).
This phenomenon was further confirmed by the analysis of the expression of osteoclastogenic genes, including NFATc1, c-Fos, and ATP6V0D2 (Fig. 4c). Furthermore, M-CSF-induced phosphorylation of MAPKs (ERK, JNK, and p38) and Akt was blocked by heparan sulfate but not chondroitin sulfate (Fig. 4d). These findings suggest that the inhibition of osteoclast precursor proliferation and differentiation by heparan sulfate may be associated with M-CSF malfunction. To determine the causative factor responsible for the defect in M-CSF function, we performed a competition binding assay. The binding of syndecan-1 to 4 ectodomains to M-CSF was significantly inhibited by heparan sulfate, but not by chondroitin sulfate, in a concentration-dependent manner (Fig. 4e). Also, the binding of syndecan ectodomains to VN and OPN was inhibited by heparan sulfate, suggesting an interaction between heparan sulfate and extracellular matrix proteins ( Supplementary Fig. 5). Overall, these results indicate that heparan sulfate chains attached to syndecan ectodomains are associated with the negative regulation of osteoclastogenesis through direct binding to M-CSF and extracellular matrix proteins.

2-O and 6-O-sulfate groups of heparin are critical for the inhibition of osteoclast differentiation
Despite the inhibitory action of heparan sulfate in osteoclastogenesis, its clinical use in bone repair may be restricted owing to its anticoagulant activity. We observed that heparan sulfate exhibited anticoagulant activity in a dose-dependent manner ( Supplementary  Fig. 6). Heparin was reported to be chemically related to heparan sulfate, displaying the same disaccharide backbone 11 . It is well established that both heparan sulfate and heparin could equally activate FGF signaling by binding to FGFs and their receptors 31,32 . Moreover, desulfated heparin derivatives showed no detectable anticoagulant activity as compared with heparin 33,34 . Based on these facts, we investigated the effects of various desulfated heparin derivatives on osteoclast precursor proliferation and differentiation. N-desulfated heparin with no anticoagulant activity ( Supplementary Fig. 6) showed higher inhibition of M-CSF-dependent osteoclast precursor proliferation than 2-O or 6-O-desulfated heparin (Fig. 5a, upper panel); we failed to observe this phenomenon in M-CSF-independent RAW264.7 cells (Fig. 5a, lower panel). In addition, N-desulfated heparin markedly suppressed osteoclast differentiation as compared to 2-O and 6-O-desulfated heparin (Fig. 5b), with a concurrent decrease in the expression of osteoclastogenic genes as well as M-CSF-induced osteoclastogenic signaling (Figs. 5c, d). To test the interaction between various desulfated forms of heparin and M-CSF, His-tagged syndecan ectodomains were immobilized on a Ni-coated plate and incubated with M-CSF and various forms of desulfated heparin. N-desulfated heparin strongly restrained the binding between syndecan ectodomains and M-CSF as compared to 2-O-or 6-Odesulfated heparin (Fig. 5e). Also, N-desulfated heparin inhibited binding of syndecan ectodomains to VN and OPN, supporting the notion that N-desulfated heparin interacts with extracellular matrix proteins ( Supplementary Fig. 7). Our findings demonstrate that N-desulfated heparin with 2-O and 6-O-sulfated groups and no anticoagulant activity in blood may inhibit osteoclastogenesis by binding to M-CSF and extracellular matrix proteins.

Heparan sulfate and N-desulfated heparin inhibit osteoclast formation and bone resorption in vivo
As shown in Fig. 4 and Fig. 5, we observed that heparan sulfate and N-desulfated heparin inhibited osteoclast differentiation in vitro. We, therefore, analyzed the effect of heparan sulfate and N-desulfated heparin on osteoclast formation in vivo. The administration of heparan sulfate and N-desulfated heparin to calvarial periosteum of mice daily for 5 days resulted in a decrease in TRAP-positive mature osteoclast formation on the surface of the calvarial bone by~50 and~32% relative to control, respectively (Fig. 6a). In addition, the histological analysis of calvarial bone sections stained with H&E revealed that the area of calvarial bone marrow cavity, which represents  Fig. 4e. Results represent the means ± SD (n = 4 as in a; n = 3 as in b and e). The p value indicates the comparison between the treatment group and control. *p < 0.05; **p < 0.01 osteoclastic bone resorption activity, was significantly decreased in heparan sulfate and N-desulfated heparinchallenged mice as compared to control mice (Fig. 6b). Consistent with this, the bone-resorptive activity of mature osteoclasts in vitro was significantly reduced by treatment with ectodomains of syndecan-1, 2, and 4 from HEK293E cells, heparan sulfate, and N-desulfated heparin compared to the controls (Supplementary Fig. 8). Thus, heparan sulfate and N-desulfated heparin negatively regulated osteoclast differentiation in vivo, thereby suppressing osteoclastic bone resorption.

Discussion
This is the first study to demonstrate that syndecan ectodomains originated from mammalian cells, heparan sulfate chains, and N-desulfated heparin derivatives could directly interact with M-CSF and decrease osteoclast formation in vitro and in vivo. This inhibitory effect was attributed to the functional blocking of M-CSF-mediated osteoclastogenic signaling. Syndecan ectodomains, heparan sulfate chains, and N-desulfated heparin are thought to play a role as a soluble decoy receptor for M-CSF and may compete with M-CSF receptor, c-Fms, for binding to M-CSF in the extracellular compartment. Some decoying factors for M-CSF may disturb the osteoclastogenic signaling via M-CSF-mediated c-Fms axis. We also found that syndecan ectodomains directly bind to extracellular matrix proteins. Thus, the combined results show that the shed ectodomains of syndecans bound to extracellular matrix proteins recruit M-CSF via heparan/chondroitin sulfate side chains and the defective M-CF signaling caused by the decreased binding opportunity between M-CSF and c-Fms retards osteoclast differentiation.
Syndecan-1 ectodomains derived from tumor cells have been reported to exhibit opposite effects on osteoclastogenesis. Syndecan-1 ectodomain purified from multiple myeloma cells significantly inhibited osteoclast differentiation at picomolar concentrations 26 . In contrast, syndecan-1 ectodomains in conditioned medium from breast cancer cells played a critical role in the stimulation of osteoclastogenesis in human peripheral blood mononuclear cells 18 . Our findings showed that murine ectodomains of syndecan-1 to 4 expressed from human embryonic kidney cells inhibited the differentiation of murine bone marrow-derived osteoclast precursors. We and others have demonstrated that heparan sulfate chains of syndecan ectodomains are essential for osteoclastogenesis regulation, but these results were controversial as described above. Heparan sulfate chains of syndecan ectodomains have been found to exhibit molecular polymorphism owing to different post-translational modifications. Thus, heparan sulfate chains with distinct functions could be produced in a specific cell or tissue type during different physiological states, neoplastic transformation, and lung development 19,[35][36][37] . Tumor heparan sulfate chains were reported to differ in their sizes, fine structures, and ligand affinities from heparan sulfate chains produced in the corresponding normal tissue 9 . Thus, the controversial effects of syndecan ectodomains on osteoclastogenesis may be explained through the structural polymorphism of their heparan sulfate chains.
It has been reported that the ectodomain of the mouse syndecan-1 core protein produced in bacteria inhibited the spreading and migration of human breast carcinoma cells through the formation of a signaling complex with integrin αvβ3 38 . Syndecan-1 core protein was reported to contain extracellular regulatory sites required for its interaction with integrin αvβ3 39 . In osteoclasts, integrin αvβ3 plays a critical role in controlling cell adhesion, migration, and differentiation 40,41 , suggesting that bacterially expressed core proteins of syndecan ectodomains may participate in the regulation of osteoclast differentiation through the interaction with, and activation of, integrin αvβ3. In addition, we observed that bacterially produced syndecan ectodomains lacking heparan sulfate chains inhibited osteoclast differentiation. This inhibitory effect of core proteins of syndecan ectodomains is thought to regulate osteoclastogenesis regardless of M-CSF.
Heparin, one of the most important anticoagulant drugs in the clinic for a long time, inhibits tumor development and metastasis and limits several inflammatory diseases 11,42,43 . The clinical use of heparin in the development of drugs for inflammatory diseases and cancers may be limited by its anticoagulant activity 43 . Thus, the anticoagulant activity should be considered for the development of heparin as a drug. Selectively desulfated forms of heparin were reported to retain the antiinflammatory and anti-tumor functions with minimal anticoagulant effects 33,34,44 . In this study, we revealed that selectively N-desulfated heparin with no detectable anticoagulant activity may be useful for anti-osteoporotic therapy, reducing the risk of unwanted hemorrhagic side effects.
Sulfation was known to play a critical role in the biological activities of heparin and heparan sulfate and regulates their binding to various proteins and growth factors through electrostatic interaction 45,46 . In heparin and heparan sulfate, sulfation mainly occurs at N, 2-O, and 6-O-positions 47,48 and growth factors recognize specific sulfate groups 49 . For instance, 2-O-sulfate groups of heparin were essential for the binding to FGF-2, and 6-Osulfate groups mediate binding to the receptor, FGFR-1 50 . The 6-O-sulfate groups of heparan sulfate are necessary for the interaction with HGF 51 and the binding affinity of 2-O-desulfated heparin for bone morphogenetic protein-2 was greater than that of heparin 52 . In line with the fact highlighting the distinct role of specific sulfate groups of heparin/heparan sulfate in binding to cytokines or growth factors, our study showed that N-desulfated heparin with 2-O-and 6-O-sulfate groups interacts with M-CSF and consequently suppress osteoclast differentiation. Osteoprotegerin (OPG), a soluble decoy receptor for RANKL, was shown to bind to heparin and heparan sulfates 53 . Heparin was reported to indirectly enhance osteoclast differentiation via binding to OPG secreted by osteoblasts 54 . Intact heparin and 2-O-desulfated heparin may bind to OPG and stimulate osteoclastogenesis, but N-desulfated heparin showed no interaction with OPG and failed to activate osteoclastogenesis, indicative of the important role of N-sulfate groups of heparin in osteoclast differentiation 54 . Considering the broad range of heparinbinding proteins, it is not surprising that intact heparin may interact with both M-CSF and OPG to exert opposite effects on osteoclastogenesis. As the anticoagulant activity of intact heparin limits its clinical use, selectively desulfated heparin derivatives with no anticoagulant effects may offer advantages for targeting metabolic bone diseases. Based on our results, N-desulfated heparin that suppresses osteoclast differentiation and osteoclastic bone resorption (via binding to M-CSF) may be used as a therapeutic agent for osteoporotic bone loss. On the other hand, 2-O-desulfated heparin that stimulates osteoclastogenesis via binding to OPG may be useful for the treatment for osteopetrosis or serve as a potential drug to halt the progression of osteopetrosis. As heparin and heparan sulfate were reported to inhibit osteoclastogenesis in RAW 264.7 cells in the absence of M-CSF and OPG 55 , we suggest that heparin and heparan sulfate chains may interact with other factors involved in the regulation of osteoclast differentiation.
In conclusion, we showed that syndecan ectodomains, heparan sulfate, and N-desulfated heparin exert suppressive effects on osteoclastogenesis and osteoclastic bone resorption. In particular, selectively N-desulfated heparin derivatives may be clinically useful as anti-resorptive agents for osteoporosis without side effects associated with the anticoagulant action.

Construction, expression, and purification of syndecan ectodomains
For bacterial expression of mouse syndecan ectodomains, the cDNAs for syndecan ectodomains were obtained by polymerase chain reaction (PCR) using primers listed in Supplementary Table 1. The PCR products were digested with NheI/NotI and inserted into the corresponding sites of pET28a vector. Escherichia coli Rosetta 2 (DE3) cells (Novagen, Darmstadt, Germany) were transformed with the pET28a-syndecan ectodomain constructs. The expression of syndecan ectodomains was induced with the addition of 0.25 mM isopropyl-β-Dthiogalactopyranoside (BioBasic Inc., Amherst, NY, USA) at 25°C for 18 h. Harvested cells were lysed in a lysis buffer (20 mM Tris-HCl, pH 7.5, and 1 mg/mL lysozyme) by sonication and the cell lysates were bound to Ni-NTA agarose beads (ELPIS Biotech). The beads were washed twice with a washing buffer [20 mM Tris-HCl, pH 7.5, 0.5 M sodium chloride (NaCl), and 60 mM imidazole] and eluted with an elution buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, and 0.5 M imidazole). The eluted proteins were desalted and exchanged with phosphate-buffered saline (PBS) using ultrafiltration system (10-kDa cut-off; PALL Life Sciences, Port Washington, NY, USA). For the mammalian expression of mouse syndecan ectodomains, the cDNAs of syndecans were amplified by PCR using primers listed in Supplementary Table 2. The PCR products were digested with EcoRI/NotI and ligated into the correct reading frames of the mammalian expression vector pIRES. HEK293E cells were transfected with pIRES-syndecan ectodomain vectors and cultured with DMEM supplemented with 1% FBS and 2 μg/mL puromycin (AMRESCO, Solon, OH, USA). The culture medium was collected every other day for 2 weeks. The collected media were concentrated and exchanged with a buffer containing 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, and 10 mM imidazole using ultrafiltration. After incubation with Ni-NTA agarose beads, the beads were washed and eluted with an elution buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, and 0.5 M imidazole). The purified recombinant proteins were concentrated and dialyzed with PBS using ultrafiltration.

Osteoclast differentiation and bone pit formation
Bone marrow-derived cells were collected from the femurs and tibias of 6-week-old C57BL6 male mice (Central Lab Animal; Seoul, Korea) by flushing the bone marrow. Cells were treated with a red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The remaining cells were incubated with α-MEM supplemented with 10% FBS and M-CSF (5 ng/mL) for 12 h. Floating cells were harvested and cultured with M-CSF (30 ng/mL) for 3 days to generate osteoclast precursors. Osteoclast differentiation was induced by culturing osteoclast precursors with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. Additionally, RAW264.7 cells were induced to differentiate into osteoclasts by treatment with RANKL (100 ng/mL) for 4 days. TRAP staining was performed using a leukocyte acid phosphatase staining kit (Sigma-Aldrich) as per the manufacturer's protocol and TRAP-positive multinucleated cells [TRAP(+) MNCs] with more than three nuclei were counted using a light microscope. For TRAP activity assay, osteoclast precursors (4 × 10 4 cells/well) were seeded into a 24-well culture plate and cultured with M-CSF and RANKL for 2 days. The cells were lysed with TRAP buffer (120 mM sodium acetate, pH 5.2, and 12 mg/mL sodium tartrate) containing 1% Triton X-100. After centrifugation, the supernatant was mixed with pnitrophenyl phosphate solution (Sigma-Aldrich) and incubated at 37°C for 30 min. The reaction was stopped by adding 1 N sodium hydroxide (NaOH) and the absorbance was measured at 405 nm wavelength using a microplate reader (Bio-Rad, Hercules, CA, USA). For the bone resorption assay, mature osteoclasts (0.5 × 10 3 cells/well in a 96-well culture plate) were detached from the culture dish using a cell dissociation solution (Sigma-Aldrich), seeded onto dentine slices (Immunodiagnostic Systems Ltd., Boldon, UK) and further incubated with α-MEM containing M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 2 days to allow bone resorption. Cells were then removed from the dentine slices by ultrasonication, and the slices were stained with hematoxylin (Sigma-Aldrich). The area of resorbed pits was analyzed using a light microscope and Image-Pro Plus version 6.0 software (MediaCybermetics, Silver Spring, MD, USA).

Measurement of anticoagulant activity
The anticoagulant activity of heparan sulfate or heparin derivatives was measured using the Anti-Xa heparin test kit (Iduron, Cheshire, UK) according to the manufacturer's protocol. Heparan sulfate (Sigma-Aldrich) or desulfated heparin derivatives (Iduron) were incubated with anti-thrombin solution (0.5 IU/mL) for 2 min at 37°C . Cells were treated with Factor Xa (2.5 μg/mL) for 2 min at 37°C. Factor Xa substrate (0.625 mg/mL) was added to develop color and the reaction was stopped with 20% acetic acid. The absorbance was measured at 405 nm using a microplate reader (Bio-Rad).

In vitro binding assay
For the analysis of the interaction between syndecan ectodomains and M-CSF using a pull-down method, recombinant human M-CSF (1 μg, 146 amino acid residues between amino acids 36 and 181) was incubated with His-syndecan ectodomains (1 μg) immobilized on Ni-NTA agarose beads (ELPIS-Biotech) in a binding buffer [20 mM Tris-HCl, pH 7.3, 150 mM potassium chloride (KCl), 0.2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol, 20% glycerol, 0.1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride] at 4°C for 12 h. The beads were washed five times using a binding buffer and centrifuged. M-CSF present in the pellet was detected by immunoblot analysis with a specific antibody against M-CSF.
For the evaluation of the interaction between syndecan ectodomains and extracellular matrix proteins, 96-well culture plates were coated with VN (10 μg/mL, BD Biosciences, San Jose, CA, USA) or OPN (10 μg/mL, Sigma-Aldrich) at 4°C for 12 h. The coated plates were blocked with the binding buffer (20 mM Tris-HCl, pH 7.3, 150 mM NaCl, 0.1% BSA, and 0.05% Tween-20) for 1 h at room temperature and washed with the washing buffer. The plates were incubated with His-tagged syndecan ectodomains from HEK293E cells (10 pmol/well) for 2 h at room temperature and sequentially exposed to anti-His antibody, HRP-labeled secondary antibody, and substrate. The binding affinity was analyzed by measuring the absorbance at 450 nm wavelength.
For the competition binding assay, a mixture of M-CSF (20 pmol/well) and heparan sulfate, chondroitin sulfate (Sigma-Aldrich), or desulfated heparin derivatives was incubated with His-tagged syndecan ectodomains (10 pmol/well) bound onto Ni-coated 96-well plates for 2 h at room temperature. To detect M-CSF bound to His-tagged syndecan ectodomains immobilized on Ni-coated plates, the plates were washed and incubated with anti-M-CSF antibody and HRP-labeled anti-rabbit IgG. The plates were washed five times and developed with TMB substrate (Pierce). The reaction was stopped by adding 5 N sulfuric acid (H 2 SO 4 ) and the absorbance was read at 450 nm using a microplate reader (Bio-Rad).
Animal studies C57BL6 male mice were purchased from Central Lab Animal (Seoul, Korea) and maintained at the animal facility of Yeungnam University College of Medicine. All animal experiments were approved by the institutional review board of Yeungnam University Medical Center and were in compliance with the Guide for the Care and Use of Laboratory Animals. Mice at 8 weeks of age were subcutaneously injected with ectodomains of recombinant mouse syndecan-1 to 4 (0.2 mg/kg in 100 μL PBS), heparan sulfate (10 mg/kg in 100 μL PBS), heparin derivatives (10 mg/kg in 100 μL PBS), or PBS alone (control) over the calvaria every day for 5 days. After 4 days, mice were euthanized and calvaria were collected for the evaluation of osteoclast formation and bone cavity. The specimens fixed with formaldehyde were embedded in paraffin and sectioned. Paraffin-embedded sections of calvaria were deparaffinized and stained with TRAP to analyze osteoclast formation, and H&E staining was performed to assess bone marrow cavity. To measure bone cavity, images were scanned with an Aperio ScanScope Model T3 and analyzed with ImageScope software (Aperio Technologies, Vista, CA, USA).

Statistical analysis
All data are presented as mean ± standard deviation (SD) from at least three independent experiments. Data with more than three groups were evaluated using analysis of variance, followed by Bonferroni's comparison test between two groups with GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). For all experiments, p < 0.05 indicated significance.