Lithium chloride modulates chondrocyte primary cilia and inhibits Hedgehog signaling

Lithium chloride (LiCl) exhibits significant therapeutic potential as a treatment for osteoarthritis. Hedgehog signaling is activated in osteoarthritis, where it promotes chondrocyte hypertrophy and cartilage matrix catabolism. Hedgehog signaling requires the primary cilium such that maintenance of this compartment is essential for pathway activity. Here we report that LiCl (50 mM) inhibits Hedgehog signaling in bovine articular chondrocytes such that the induction of GLI1 and PTCH1 expression is reduced by 71 and 55%, respectively. Pathway inhibition is associated with a 97% increase in primary cilia length from 2.09 ± 0.7 μm in untreated cells to 4.06 ± 0.9 μm in LiCl‐treated cells. We show that cilia elongation disrupts trafficking within the axoneme with a 38% reduction in Arl13b ciliary localization at the distal region of the cilium, consistent with the role of Arl13b in modulating Hedgehog signaling. In addition, we demonstrate similar increases in cilia length in human chondrocytes in vitro and after administration of dietary lithium to Wistar rats in vivo. Our data provide new insights into the effects of LiCl on chondrocyte primary cilia and Hedgehog signaling and shows for the first time that pharmaceutical targeting of the primary cilium may have therapeutic benefits in the treatment of osteoarthritis.—Thompson, C. L., Wiles, A., Poole, C. A., Knight, M. M. Lithium chloride modulates chondrocyte primary cilia and inhibits Hedgehog signaling. FASEB J. 30, 716‐726 (2016). www.fasebj.org

Lithium chloride, a drug previously used for the treatment of bipolar disorder (1)(2)(3), has recently been shown to exhibit significant therapeutic potential as a treatment for arthritis (4,5). These studies show that LiCl inhibits cartilage degradation in vitro in response to the inflammatory cytokine IL-1. LiCl disrupts the downstream activation of p38 MAPK and nuclear factor k-light-chain-enhancer of activated B cells (NF-kB) in response to IL-1, thus inhibiting the induction of catabolic enzymes such as A disintegrin and metalloproteinase with thrombospondin motifs 5 and matrix metalloproteinases 3 and 13. Consequently, this inhibits cartilage degradation (4,5) and loss of cartilage mechanical properties (6). Similar findings have been reported in vivo wherein both dietary and intra-articular injection of LiCl were found to reduce the severity of cartilage damage in surgical models of osteoarthritis (OA) (5).
Recent studies have shown that LiCl inhibits Hedgehog signaling in pancreatic ductal adenocarcinoma cells (7). Several studies have now implicated Hedgehog signaling in the development and progression of OA (8)(9)(10)(11)(12). The expression of Indian hedgehog (Ihh) is increased in early cartilage lesions (8,12), and the concentration of Ihh in the synovial fluid increases with the severity of cartilage damage in human knee joint OA (11,12). Consequently, Hedgehog signaling is activated in osteoarthritic cartilage, where it promotes chondrocyte hypertrophy and matrix catabolism (10,11). In mouse models, pharmacologic inhibition of the Hedgehog pathway reduces the severity of surgically induced OA (10). More recent studies have reported that inducible, cartilage-specific deletion of Ihh also prevents disease progression, highlighting the importance of ligand-mediated signaling in this disease (9).
LiCl has been shown to affect the primary cilium, a dedicated signaling compartment at the cell surface (13)(14)(15)(16). The primary cilium is rapidly becoming recognized as an important player in arthritis as a result of its established roles in Wnt (17,18) and Hedgehog signaling (19), and more recently in its involvement in chondrocyte mechanotransduction (20)(21)(22). The cilium comprises a modified centriole called the basal body from which extends a microtubule-based scaffold, the ciliary axoneme. The axoneme is surrounded by a distinct membrane, which can become specialized in a cell-type-specific manner. A gated diffusion barrier exists at the ciliary base, and thus the entry and exit of proteins into the ciliary axoneme is tightly regulated (23,24). Soluble proteins and transmembrane receptors are conducted along ciliary microtubules by a process called intraflagellar transport (IFT) (25,26). The anterograde motor kinesin 2 is responsible for the transport of IFT cargo, like tubulin, from the base of the cilium to the distal tip, while cytoplasmic dynein 2 mediates retrograde transport from the tip to base (27,28). Cilia assembly and disassembly is initiated at the distal tip; IFT is thus essential to maintain the size of the ciliary compartment (29). Studies have shown that LiCl promotes significant elongation of the ciliary axoneme in fibroblasts (14,15) and neuronal cells (13); however, the consequences of this elongation for cilia-mediated signaling pathways has not been explored.
The coordinated transport of Hedgehog signaling components through the ciliary compartment is essential for pathway activation. The Hedgehog receptor Patched (Ptch1) is localized to the ciliary membrane in the absence of ligand. Upon ligand binding, Ptch1 exits the cilium and triggers the ciliary accumulation of a second transmembrane protein, Smoothened (Smo), a process that is mediatedbyanumberofproteinssuchasthesmallGTPase Arl13b (30,31). Within the cilium, Smo regulates the activity of the Gli transcription factors (Gli2 and Gli3), the downstream effectors in this pathway (32). These transcription factors are constitutively expressed and accumulate at the ciliary tip in response to ligand; once activated, they translocate to the nucleus where they regulate gene transcription (19,33,34). At the nucleus, their function is further amplified by the induction of a third transcription factor, Gli1. Gli1 has also been shown to up-regulate Ptch1 expression, thereby establishing a negative-feedback mechanism (35). The measurement of both GLI1 and PTCH1 mRNA can be used to monitor pathway activity. The size, frequency, and velocity of IFT particles within the cilium are influenced by cilia length (36). This affects the delivery of cargo to the ciliary tip such that longer cilia have a reduced level of protein entry, which undoubtedly influences ciliamediated signaling (37,38). Indeed, Cruz et al. (39) report that during embryonic development, Foxj1 mediated lengthening of primary cilia in the neural tube attenuates Hedgehog signaling to regulate cellular identity and formation of the embryonic floor plate.
In the current study, we hypothesized that LiCl influences chondrocyte Hedgehog signaling and that this is achieved through its effects on primary cilia structure. We demonstrated that LiCl treatment results in the inhibition of ligand-mediated Hedgehog signaling. LiCl treatment results in rapid cilia axoneme growth in isolated bovine chondrocytes, which correlates with Hedgehog pathway inhibition and is associated with reorganization of the ciliary compartment and disruption of Arl13b ciliary localization. Furthermore, we report that LiCl induces cilia elongation in human articular chondrocytes in vitro and in vivo using a rat model designed to mimic long-term lithium treatment in human patients. Thus, we show for the first time that LiCl suppresses Hedgehog signaling through alterations in primary cilia structure.
Human articular chondrocytes were obtained commercially (Articular Engineering, Northbrook, IL, USA). Cells were isolated from full-depth cartilage slices removed from the femoral condyles of male cadaver donors aged 60 to 71 yr. Chondrocytes were cultured in chondrocyte culture medium (Articular Engineering) at 37°C and 5% CO 2 and used in experiments from P0 to P3.
For experiments, articular chondrocytes were seeded onto serum-coated glass coverslips or plastic tissue culture plates. Upon reaching confluence, cells were treated with 0 to 50 mM LiCl (Sigma-Aldrich) for up to 24 h in the presence or absence of 1 mg/ml recombinant Indian hedgehog (r-Ihh; R&D Systems, Minneapolis, MN, USA). At the end of the experiment, cell viability was assessed by calcein AM/ethidium homodimer (Life Technologies, Carlsbad, CA, USA) labeling and fluorescence microscopy, and separate samples were processed for immunofluorescence, Western blot analysis, and real-time PCR.

Animal studies
Male Wistar rats (3 per treatment group) were used to examine the influence of dietary lithium on primary cilia length in vivo. Tissue was obtained from rats fed a lithium-enriched diet, 60 mmol lithium/100 g ground rat food, for 9 mo. Rats were initially given food containing 40 mmol lithium for the first 7 d, followed by 9 mo at the higher dose, enabling them to make physiologic adjustments to the treatment regimen. This is equivalent to approximately 20 yr of treatment in human patients, and it resulted in plasma lithium levels comparable to therapeutic levels in human plasma (0.8-1.3 mM). Plasma lithium levels were monitored through a blood sample taken from the tail vein at the end of the experimental period. Age-matched controls were kept on normal food and water. To minimize the lack of weight gain associated with lithium-induced diuresis, rats were given continuous access to a salt block and free access to water. These studies were approved by the Otago University animal ethics committee (AEC 98/10).

Immunofluorescence confocal and super resolution microscopy
For isolated cells, coverslips were fixed with 4% paraformaldehyde for 10 min. Fixation was followed by detergent permeabilization with 0.5% Triton X-100 and blocking with 5% serum for 1 h. Samples were incubated with primary antibodies at 4°C overnight. After repeated washing in 0.1% bovine serum albumin/PBS, cells were incubated with appropriate Alexa Fluor-conjugated secondary antibodies (1:1000; Life Technologies) for 1 h at room temperature and nuclei were detected with 1 mg/ml DAPI. After repeated washing in 0.1% bovine serum albumin/PBS, coverslips were mounted with Prolong gold reagent (Life Technologies).
For immunohistochemical staining of primary cilia, cartilage samples were fixed in 10% neutral buffered formalin for 48 h, decalcified in 10% EDTA pH 7 0 to 7.2, and embedded in paraffin wax. Sections 20 mm thick were cut perpendicular to the surface of the cartilage; paraffin was then removed and samples were processed as above for the detection of primary cilia.
For primary cilia length measurement, samples were imaged using a Leica TCS SP2 confocal microscope with a 363, 1.3 NA lens. Confocal Z stacks were generated throughout the entire cellular profile using a Z step size of 0.5 mm, 32zo o m ,a nda n im a g eforma tof10 2 43 1024 pixels. This produced an xy pixel size of 0.116 3 0.116 mm. Z stacks were reconstructed and an xy maximum intensity projection used for the measurement of cilia length using ImageJ software (Image Processing and Analysis in Java; U.S. National Institutes of Health, Bethesda, MD, USA). The length of approximately 100 cilia was measured over 5 representative fields of view for each condition per experiment. For primary cilia prevalence and Ki-67 staining, samples were imaged using a Leica DMI4000B epifluorescent microscope with a 363/ 1.4 NA objective. The proportion of ciliated/Ki-67-positive cells was determined in 10 representative fields of view for each condition per experiment. For structural illumination microscopy, samples were imaged on a Zeiss 710 ELYRA PS.1 microscope with a 363/1.4 NA objective.

Western blot analysis
Cells were lysed in NP-40 buffer (1% NP-40, 150 mM NaCl, 50 mM Tris pH 8.0) containing 50 mM NaVO 4 and protease inhibitor cocktail (Roche, Basel, Switzerland). The lysate was homogenized and centrifuged at 13,000 rpm for 15 min. The supernatant was collected and protein concentration determined by bicinchoninic acid assay. SDS-PAGE was performed using 40 mg total protein and run on 5-20% gradient gels (Bio-Rad, Hercules, CA, USA). Proteins were transferred to a nitrocellulose membranes and blocked for 1 h at room temperature in 5% nonfat milk or 5% bovine serum albumin in Tris-buffered saline according to the manufacturer's recommendations, then incubated with primary antibodies at 4°C overnight. Membranes were washed and then incubated with appropriate infrared secondary antibodies (Li-Cor Biosciences, Lincoln, NE, USA) for 1 h at room temperature. Membranes were washed again and visualized using the Li-Cor Odyssey. Analysis of band intensity was performed by Li-Cor software.
RNA extraction, cDNA synthesis, and real-time PCR Total RNA was isolated from individual wells using an RNeasy Kit (Qiagen, Germantown, MD, USA) and converted to cDNA using the Quantitect reverse transcription kit (Qiagen) according to the manufacturer's instructions. For real-time PCR, reactions were performed in 10 ml volumes containing 1 mlcDNA(diluted1:2), 5 ml Kapa SYBR Fast Universal 23 qPCR Master Mix (Kapa Biosystems, London, United Kingdom) containing SYBER green dye, 0.2 ml ROX reference dye, and 1 ml optimized primer pairs ( Table 1). An annealing temperature of 60°C was used for all PCR reactions. Fluorescence data was collected using the MX3000P qPCR instrument (Stratagene) and analyzed using the relative standard curve method (42).
The expression of GLI1 and PTCH1 were monitored as a measure of Hedgehog pathway activity and normalized to the expression of 18S ribosomal RNA, which was not significantly altered by the experimental conditions used in this study.

Statistical analyses
All statistical analyses were conducted by Graph Pad Prism 6.01 (GraphPad, La Jolla, CA, USA). N refers to the number of donors (independent experiments) and n refers to the number of technical replicates analyzed per experimental group. Data are presented as means 6 SD, except for cilia length, for which median values are described. Before analyses, data sets that failed normality testing (Shapiro-Wilk test) were converted to a Gaussian distribution by Box-Cox transformation. Thereafter, all data were analyzed by 1-or 2-way ANOVA and as indicated in the figure legends with post hoc Tukey's multiple comparisons. Differences were considered statistically significant at P # 0.05.

Ligand-dependent Hedgehog signaling is inhibited by LiCl
After 24 h r-Ihh treatment, real-time PCR analysis showed that the mRNA levels of GLI1 (Fig. 1A) and PTCH1 (Fig. 1B) were significantly increased by 4.48and 3.16-fold, respectively. LiCl treatment alone did not significantly affect gene expression. However, dosedependent inhibition of GLI1 and PTCH1 (r 2 = 0.8685 and 0.9043, respectively; Supplemental Fig. S1) induction in response to r-Ihh was observed such that Hedgehog pathway activation was not significantly activated in the presence of 50 mM LiCl (Fig. 1A, B). Chondrocyte viability was maintained throughout the experimental period, and effects on cell cycle status were minimal (Supplemental Fig. S2). These data indicate that LiCl inhibits ligand-dependent Hedgehog signaling in bovine articular chondrocytes.

Hedgehog pathway inhibition is independent of glycogen synthase kinase
LiCl functions primarily as a glycogen synthase kinase (GSK) inhibitor, the blockade of which results in the accumulation of b-catenin and activation of canonical Wnt signaling (43). After 24 h LiCl treatment, Western blot analysis revealed that the level of activated b-catenin was not significantly altered (Fig. 1C, D), suggesting that LiCl does not influence Hedgehog signaling through GSK inhibition and canonical Wnt signaling. Indeed, the induction of GLI1 and PTCH1 in response to r-Ihh was not significantly different in the presence of the GSK inhibitor CHIR-99021 (Supplemental Fig. S3).
The effects of 50 mM NaCl and KCl on cilia prevalence (Fig. 2D) and length (Fig. 2E)werealsoexamined. Alterations in cilia length were observed with KCl only; however, these were significantly less pronounced, suggesting that dramatic elongation is specifict oL i C l (Fig. 2D).

Primary cilia elongation is independent of GSK
Nakakura et al. (14) reported that in fibroblasts, LiClmediated inhibition of GSK releases an inhibitory block on the enzyme a-tubulin acetyl transferase 1, causing a dramatic increase in whole-cell acetylated tubulin levels and an increase in cilia length. Immunocytochemistry revealed that the distribution of acetylated tubulin was altered in chondrocytes treated with 50 mM LiCl (Fig. 3A) such that staining was increased at the ciliary base (Fig. 3A,inset).However,nosignificant differences in the total levels of tubulin acetylation were identified by Western blot analysis (Fig. 3B, C). Moreover, the GSK inhibitor CHIR-99021 did not significantly influence cilia prevalence or length (Fig. 3D-F), suggesting that cilia elongation occurs through an alternative mechanism in articular chondrocytes.

Primary cilia elongation is rapid, occurring in 1 h of LiCl treatment
T h et e m p o r a le f f e c t so fL i C lo nc i l i al e n g t hw e r ea n alyzed over 1 to 24 h. In control cells, cilia length did not vary significantly over the 24 h period (Supplemental Fig. S4). In response to LiCl, cilia length was significantly increased after 1 h at all doses ( Fig. 4). At 1 mM LiCl, cilia length was increased at 1 and 3 h; however, this response was not maintained over 24 h (Fig. 4A). In contrast, maximal cilia elongation was achieved at 1 h in response to 10 mM LiCl and was maintained throughout the culture period (Fig. 4B). At 50 mM LiCl, the axoneme continued to grow after 1 h, but at a slower rate, reaching a median length of 4.06 6 0.9 mmb y2 4h (Fig. 4C). These data indicate that ciliary elongation in vitro is rapid, but transient at low doses.

LiCl-induced cilia elongation is associated with reorganization of ciliary compartment
Arl13b, a ciliary protein within the ADP-ribosylation factor family and Ras superfamily of GTPases, has been implicated in the dynamic localization of Hedgehog signaling components to the ciliary compartment in response to ligand stimulation (30). After 1 h LiCl treatment, the proportion of primary cilia exhibiting accumulations of Arl13b at their distal tip was increased, giving the appearance of a swollen or bulbous tip (r 2 = 0.5509, Fig. 5A and Supplemental Fig. S1). In response to 50 mM LiCl 15.3 6 8.6% of cells exhibited bulbous tips compared with just 4.5 6 0.81% in untreated cells (Fig. 5B). After 24 h, the proportion of cilia with swollen tips had returned to the level of the control (data not shown). These tip structures were observable using structural illumination microscopy, which also highlightsthemembranelocalizationofthisprotein,which becomes increasingly patchy and disrupted along the length of the axoneme as cilia elongate (Fig. 5C). The presence of bulbous tips is generally associated with defects in retrograde IFT; thus, these data are consistent with a dose-dependent imbalance in the molecular transport mechanisms regulating cilia length (48,49).
To examine the effects of LiCl on Arl13b trafficking, intensity measurements were taken along the length of the axoneme. At 1 h, the Arl13b intensity profile appeared relatively uniform in control cells and cells treated with 50 mM LiCl (Fig. 5D, E). Moreover, mean Arl13b intensity was not significantly different between the 2 groups (Fig. 5I). In contrast, while the Arl13b intensity profile remained relatively uniform in control cilia at 24 h ( Fig. 5F), in cells treated with 50 mM LiCl Arl13b, intensity appeared weaker toward the distal tip (Fig. 5G), and mean Arl13b intensity was significantly reduced in these cilia (Fig. 5J). Division of the cilium into proximal and distal regions confirmed that this was largely due to a reduction in the distal region at 24 h (Fig. 5I, K).
At both 1 and 24 h, total Arl13b staining within the axoneme was increased, suggestive of an increase in ciliary trafficking. However, while at 1 h the amount of Arl13b per micrometer of axoneme remained constant between control and treated cilia, at 24 h this was significantly reduced, thus highlighting the possibility that a threshold length exists at which the Arl13b trafficking equilibrium can be maintained.

LiCl induces primary cilia elongation in isolated human chondrocytes and in vivo in rat chondrocytes
After 24 h treatment with 50 mM LiCl, primary cilia length was significantly increased in isolated human articular chondrocytes (Fig. 6A, B), suggesting that this phenomenon could be relevant for human patients. However, the physiologic dose of lithium in patients undergoing therapy is significantly lower than that used in the current in vitro study, ranging from 0.8 to 1.3 mmol/L. The effects of lithium on chondrocyte primary cilia length were therefore explored in vivo using a rat model. Primary cilia were labeled for acetylated a-tubulin (Fig. 6C), and primary cilia length was quantified through the depth of the articular cartilage. In rats receiving dietary lithium, primary cilia length was significantly increased relative to the control group from 1.44 6 0.6 to 1.72 6 0.6 mm, respectively, indicating that long-term treatment with physiologically relevant doses can influence primary cilia structure in vivo (Fig. 6D).

DISCUSSION
The current study demonstrated that LiCl induces primary cilia elongation in articular chondrocytes both in vitro and in an in vivo rat model. In the presence of LiCl, liganddependent Hedgehog signaling was significantly inhibited, directly correlating with these changes in cilia length. We suggest that this inhibition may be related to the reorganization of the ciliary compartment and dilution of Hedgehog signaling components such as Arl13b (Fig. 5J) within the axoneme. Mahjoub and Stearns (50) report that in cells exhibiting supernumerary centrioles, the multiciliated phenotype that arises inhibits Hedgehog signaling. Dilution of Hedgehog signaling components, such as  . Primary cilia length in untreated cells did not vary significantly over 24 h period; thus, these data are presented as single box-and-whisker plots for clarity (Con). For box-and-whisker plots, line is median, box 25th to 75th percentile with 10th to 90th percentile whiskers. Statistical analysis performed by 2-way ANOVA with Tukey's multiple comparisons tests. Statistical significance is expressed relative to control data set for each time point (Supplemental Fig. S4). *P , 0.05, ***P , 0.001.
Arl13b and Smo, across multiple primary cilia reduces the amount of protein per micrometer in the axoneme and inhibits pathway activity (50), similar to what we observed for a single elongated cilium in the current study.
Mice lacking Arl13b exhibit abnormal Hedgehog signaling (31). In null mutants, Smo localizes exclusively to the ciliary compartment, and consequently Gli transcriptional activators are constitutively active, albeit at low levels Figure 5. LiCl-induced primary cilia elongation is associated with reorganization of ciliary compartment. A) Immunofluorescence for Arl13b. Arrow identifies accumulation of protein forming bulb at ciliary tip observed at 1 h. Scale bar, 10 mm. B) Prevalence of cilia with bulbous tips after 1 h LiCl treatment (N =3,n =15fields). C ) Immunocytochemistry for acetylated a-tubulin (red) and Arl13b (green); bulbous tips were imaged after 1 h LiCl treatment (50 mM) using structured illumination microscopy. Data represent means 6 SD. D-G) Distribution of acetylated a-tubulin (red) and Arl13b (green) staining within control cilia and LiCltreated cilia at 1 h (D, E) and 24 h (F, G). Scale bars, 1 mm. Graphs show fluorescence intensity along axoneme from base (proximal) to tip (distal). Data represent means 6 SEM (N =3,n = 30 cilia per group). H-K) Mean Arl13b intensity within cilia at 1h(H ) and 24 h (J )(N =3,n = 30 cilia). Mean Arl13b intensity in proximal and distal region of ciliary axoneme at 1 h (I)and24h (K)(N =3,n =30ciliape rgroup).Data represent means 6 SD. Statistical analysis performed by 2-way ANOVA with Tukey's multiple comparisons tests. Statistical significance is expressed relative to untreated control unless otherwise shown. ***P , 0.001. (31). In the absence of Arl13b, the enrichment of Gli proteins at the ciliary tip does not occur in response to ligand stimulation, and consequently the pathway cannot be further activated despite the constitutive ciliary localization of Smo (30). While we observed alterations in the ciliary localization of Arl13b after LiCl treatment, a lack of suitable antibodies coupled with the low expression of Hedgehog pathway components in adult chondrocytes precluded reliable quantification of Hedgehog signaling proteins within the ciliary compartment. However, we hypothesize that alterations to Ar113b trafficking and thus distribution within the cilium would similarly impact the dynamic localization of Smo and Gli proteins in response to Hedgehog ligand. However, several intraflagellar and ciliary proteins have been linked to the traffickingofHedgehogproteinsin addition to Arl13b, such as Kif3a (44,51), Kif7 (52), Dync2h1 (53), Ift88 (44,51), Ift80 (54), Ift25 (55), Ellis van Creveld protein and Ellis van Creveld protein 2 (56,57), Gpr161 (58), and, more recently, adenylate cyclase 5 and 6 (59), the function of which could also be influenced by LiCl. Alternatively, however, Hedgehog inhibition could also result from the disruption of proteins at the ciliary tip, such as Kif7, which have previously been shown to regulate Gli protein activation (60,61).
In contrast to previous reports (14,47), our results indicate that in articular chondrocytes cilia elongation does not occur as a consequence of GSK inhibition (Fig. 3D-F). This finding is consistent with our observation that GSK inhibition does not inhibit Hedgehog signaling and supports our hypothesis that cilia elongation is of importance. In neuronal cells, LiCl induces primary cilia elongation through the inhibition of adenylate cyclase and a reduction in cAMP levels (15).
We have previously shown that modulation of cAMP influences cilia length in articular chondrocytes (62). Therefore, it is likely that LiCl acts in a similar manner to trigger cilia elongation in chondrocytes. However, the influence of cAMP on chondrocyte cilia structure is complex, as both adenylate cyclase inhibition and exogenous cAMP induce cilia elongation in isolated chondrocytes (62). Intriguingly, mechanical loading both modulates cAMP levels and influences primary cilia length in articular chondrocytes, highlighting the potential existence of a feedback mechanism to regulate ciliary signaling (46,63). Indeed, changes in ciliary structure have been reported in mechanically compromised osteoarthritic cartilage such that the cilium elongates with increasing disease severity (64).
The accumulation of activated b-catenin in response to LiCl and subsequent activation of canonical Wnt signaling through the inhibition of GSKb is well established. Zinke et al. (65) have suggested that b-catenin accumulation may sequester activated Gli proteins away from the nucleus in response to LiCl treatment in medulloblastoma cells. However, in the current study, we did not observe significant accumulation of activated b-catenin, and GSK inhibition alone did not inhibit Hedgehog signaling. It therefore seems unlikely that LiCl is functioning primarily as a GSK inhibitor in articular chondrocytes. Indeed, previous studies have shown that while GSK inhibition alone negatively affects cartilage health, resulting in increased cartilage degradation and susceptibility to injury (66), administration of dietary lithium does not lead to cartilage degradation (5, 6). Lancaster et al. (67) report that the cilium exerts a negative influence over canonical Wnt signaling by sequestering b-catenin away from the nucleus such that in the presence of cilia, LiCl cannot activate Wnt Figure 6. LiCl causes primary cilia elongation in isolated human chondrocytes and in vivo in rat chondrocytes. A) Immunofluorescence of isolated human articular chondrocytes treated for 24 h with LiCl (50 mM). Scale bars, 20 mm. White boxes indicate enlarged representative cells shown under each condition. Scale bars, 5 mm. Primary cilia were labeled for acetylated a-tubulin (red) and arl13b (green); nuclei were counterstained with DAPI (blue). B) Primary cilia length (N =3 ,n $ 100 cilia per group) at 24 h. C ) Immunohistochemistry of rat chondrocytes in situ having been exposed to dietary lithium for 9 mo. Scale bars, 2 mm. Primary cilia were labeled for acetylated a-tubulin (green); nuclei were counterstained with DAPI (blue). D) Primary cilia length was quantified throughout depth of tissue (N =3 ,n $ 100 cilia per group). Data represent means 6 SD. For box-and-whisker plots, line is median, box 25th to 75th percentile with 10th to 90th percentile whiskers. Statistical analysis performed by 2-way ANOVA with Tukey's multiple comparisons tests. Statistical significance is expressed relative to untreated control. ***P , 0.001. signaling in mouse embryonic fibroblasts. Given that the majority of articular chondrocytes are ciliated, this may provide an explanation for the lack of b-catenin activation observed in the current study.
Both dietary and intra-articular injection of LiCl reduce cartilage degradation in the destabilization of medial meniscus model of OA while having no overt effects on cartilage health (5). Similarly, Hedgehog inhibition also reduces cartilage damage and slows disease progression in this model (10). More recent studies report that the classic Hedgehog pathway inhibitor cyclopamine attenuates in vivo inflammation and cartilage degradation in adjuvant-induced arthritis (68). Therefore, we suggest that in addition to its effectsoninflammatory signaling, the efficacy of LiCl in destabilization of medial meniscus studies may also be linked to its influence over Hedgehog signaling.
Minashima et al. (5) report that LiCl inhibits NF-kB signaling in response to IL-1 treatment. Previously we have reported a role for the cilium in NF-kB signaling such that, in the absence of cilia, NF-kB signaling is both delayed and attenuated, a consequence of which i st h a tc h o n d r o c y t e sa r eu n a b l et ou pr e g u l a t en i t r i c oxide and prostaglandin E 2 production in response to IL-1 (62,69). Therefore, an intriguing possibility is that LiCl might also be influencing inflammatory signaling through the modulation of cilia length; however, this remains to be investigated.
That cilia elongation was observed both in human chondrocytes and chondrocytes in vivo after lithium administration highlights the potential of this drug as a treatment for cartilage disease in human patients. Although an association between rheumatoid arthritis and psychiatric disorders has been postulated (70), few studies have explored any association between OA and bipolar disorder. Recently it was reported that the incidence of OA in patients with bipolar disorder was increased (71); however, this was not found to be associated with the use of mood stabilizers, and it therefore remains to be investigated whether patients undergoing lithium therapy are protected against this disease. We suggest that targeting Hedgehog signaling through the modulation of primary cilia structure may provide a novel means by w h i c ht or e d u c ec a r t i l a g ed e g r a d a t i o ni nO A .I m p o r t a n t l y , the involvement of the cilium in this response facilitates a multifaceted approach to OA therapy, as it may enable the targeting of multiple pathophysiologic pathways through a single organelle. The study is therefore the first to indicate the potential of pharmaceutical ciliotherapy for the treatment of OA.