Review
The chondrocyte primary cilium
R. Ruhlen
y
*, K. Marberry
z
ay A.T. Still Research Institute, A.T. Still University, Kirksville, MO, USA
z Department of Surgery, A.T. Still University-Kirksville College of Osteopathic Medicine, Kirksville, MO, USA
a r t i c l e i n f o
Article history: Received 10 December 2013 Accepted 7 May 2014 Keywords: Chondrocyte Cilia Mechanotransduction Osteoarthritiss u m m a r y
The presence and role of primary, or non-motile, cilia on chondrocytes has confused cartilage researchers for decades. Initial explanations attributed a vestigial nature to chondrocyte cilia. Evidence is now emerging that supports the role of the chondrocyte primary cilium as a sensory organelle, in particular, in mechanotransduction and as a compartment for signaling pathways. Early electron microscopy images depicted bent cilia aligned with the extracellular matrix (ECM) in a manner that suggested a response to mechanical forces. Molecules known to be mechanotransducers in other cell types, including integrins and proteoglycans, are present on chondrocyte cilia. Further, chondrocytes which lack cilia fail to respond to mechanical forces in the same manner that chondrocytes with intact cilia respond. From a clinical perspective, chondrocytes from osteoarthritic (OA) cartilage have cilia with different characteristics than cilia found on chondrocytes from healthy cartilage.
Objective: This review examines the evidence supporting the function of chondrocyte cilia and briefly speculates on the involvement of intraflagellar transport (IFT) in the signaling pathway of mechano-transduction through the cilium.
Conclusions: Emerging evidence suggests cilia may be a promising target for preventing and treating OA. © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Introduction
Articular cartilage is comprised of water, collagen, proteoglycan, and other non-collagenous proteins. The high water content of normal articular cartilage is maintained by the hydrophilic nature of proteoglycans within the collagen framework. Chondrocytes within the cartilage matrix are responsible for forming and main-taining the matrix. Articular cartilage is avascular, so chondrocytes are nourished by synovialfluid diffusion through the matrix by mechanical forces.
To maintain viability, chondrocytes require the right amount of mechanical stimulation: too little or too much loading leads to chondrocyte dysfunction and cartilage degeneration. In vivo, articular cartilage experiences routine loading. During walking, forces on the tibial surface are 2e3 times body weight, and peak loading is 4 times body weight1,2. Joint loading exerts direct strain
on the cartilage matrix as well as on the cells and induces hydro-static pressure,fluid shear, and changes in osmolarity as synovial fluid is pushed through the tissue3. Without loading, cartilage
health deteriorates4, but with excessive or abnormal loading, the joint deteriorates5. The degree, frequency, and duration of loading affect whether cartilage thrives or deteriorates. Chondrocytes have been shown to respond to abnormal joint loading by attempting to repair the damaged cartilagedas seen by elevated C-terminal telopeptide of type II collagen in the serum and urine6dbut eventually apoptosis will occur7, which suggests a range of
compression is necessary for optimal chondrocyte function and to maintain normal articular cartilage. In chondrocytes from osteo-arthritic (OA) cartilage, the response to compression is different. In one study, cyclic mechanical stimulation induced membrane hy-perpolarization, upregulation of the proteoglycan aggrecan, and downregulation of matrix metalloproteinase 3 in healthy chon-drocytes, responses which were absent in chondrocytes from OA cartilage8. In fact, the same mechanical stimulation induced the opposite response, membrane depolarization, in OA chondrocytes8. This hyperpolarization or depolarization response is thought to be involved in mechanotransduction8.
How chondrocytes detect and respond to compression and how the mechanotransduction pathway is altered in OA is largely un-known. A recent study has hypothesized that the primary cilium of
* Address correspondence and reprint requests to: R. Ruhlen, A.T. Still Research Institute, A.T. Still University, 800 W. Jefferson St., Kirksville, MO 63501, USA. Tel: 1-660-626-2889; Fax: 1-660-626-2602.
E-mail addresses: [email protected] (R. Ruhlen), [email protected]
(K. Marberry).
a Kirksville College of Osteopathic Medicine, A.T. Still University, 800 W. Jefferson
St., Kirksville, MO 63501, USA. Tel: 1-660-626-2663; Fax: 1-660-626-2636.
http://dx.doi.org/10.1016/j.joca.2014.05.011
the chondrocyte plays a role in the detection and transmission of mechanical stimulation9. Many factors involved in mechano-transduction have also been linked to cilia although not necessarily in chondrocyte cilia. The purpose of this review is to investigate the evidence for the role of chondrocyte cilia in mechanotransduction.
Cilia and chondrocytes
Cilia and a similar organelle, theflagellum, are well known for their role in cellular motility. In the lungs, motile cilia on epithelial cells lining the airways push mucus and foreign particles, including bacteria10. Cilia in the Fallopian tubes move the ovum toward the uterus10. The role of the primary cilium, an organelle that protrudes from most eukaryotic cells like a short antenna, has been more elusive. While only specialized cells have multiple motile cilia or a flagellum, nearly all eukaryotic cells have a non-motile primary cilium unless undergoing cell division10. The axoneme, or central skeleton, of primary cilia consists of a ring of nine pairs of micro-tubules, known as a 9þ0 pattern to distinguish it from the 9þ2 pattern of motile cilia that have an additional central pair of mi-crotubules11. Because microtubules are enriched in acetylated
a
-tubulin, cilia can be visualized with acetylateda
-tubulin antibodies12.With electron microscopy, primary cilia with the 9þ0 pattern have been observed on most chondrocytes13,14. Primary cilia are found on all murine chondrocytes and on 96% of equine chon-drocytes14, as well as on human and bovine chondrocytes15,16.
Immunostaining with acetylated
a
-tubulin identified primary cilia on chondrocytes within cartilage from bovine patellae16.For the remainder of this review, the term cilia will refer to primary or non-motile cilia, and motile cilia will be referred to with the adjective. Several theories have been proposed for the role of the primary cilium in chondrocytes.
Early theories of cilia function in chondrocytes
The primary cilium in most eukaryotic, non-motile cells, such as chondrocytes, was once thought to be a vestigial organelle with no identifiable function in normal cells17. In another theory, the
pres-ence of cilia was thought to indicate motility of chondrocytes, and the characteristic cluster and column formations were thought to be aggregates of motile chondrocytes15. However, additional studies failed to support the vestigial nature of cilia, and the dif-ference between motile and primary cilia became better under-stood such that the presence of a primary cilium is not considered to be evidence of motility. A third theory suggests that cilia regulate mitosis by sequestering the centriole, which localizes to the pri-mary cilium when not in use during mitosis17. While it is true that cells with primary cilia tend to be differentiated or in the G0phase
of the cell cycle, the role of primary cilia in the regulation of mitosis has not been pursued further.
Protein and lipid targeting, secretion, and endocytosis
The Golgi apparatus works closely with intraflagellar transport (IFT)dthe process which assembles, disassembles, and maintains ciliadto deliver lipids and proteins to the cilium18. The observation of vesicles pinching off the tip of theflagellum of unicellular algae gave rise to the idea that IFT is involved in secretion19. IFT has been identified in non-ciliated cells and is associated with exocytosis19.
Electron microscopy and confocal imaging revealed a close asso-ciation between cilia and the Golgi apparatus in aortic smooth muscle and in articular chondrocytes, leading to the proposal that the cilium is part of a sensory feedback loop which begins with mechanical transduction and ends with extracellular matrix (ECM)
protein secretion20. In addition to exocytosis and secretion, the cilia
may be involved in endocytosis. In synoviocytes, cilia reside in a dip in the membrane called the ciliary pocket21. Cilia in chondrocytes have been shown to reside in similar pockets, and the presence of clathrin-coated vesicles, endosomal proteins, and CD44 in the ciliary pocket indicate endocytosis21.
Cartilage organization and skeletal patterning
Cilia may direct the columnar organization of chondrocytes, with the columnar and cluster formations representing clonal groups22. Mutations in ciliary proteins result in developmental skeletal defects, such as digit patterning, endochondral bone, craniofacial, and dentition abnormalities12. Indian hedgehog (Ihh) and Wingless (Wnt) proteins, found in cartilage, are important in skeletal development12. Sonic hedgehog (Shh), also present in cartilage, is necessary for proper digit patterning12. Receptors for these ligands are present on chondrocyte cilia12. Therefore, ciliary defects may involve abnormal regulation of Ihh, Wnt, and Shh12.
Sensory organelle
The role of cilia in mechanotransduction stems from the obser-vation that some of the cilia in chondrocytes appear bent23. Obser-vations through transmission electron microscopy, electron tomography, and confocal microscopy allowed visualization of the interaction between cilia bending patterns and the ECM. In the absence of matrix, all cilia were straight, with a wide range of bending patterns observed in cilia from chondrocytes that were within a cartilage matrix (Fig.1). This observation suggests that cilia bending is a passive response to mechanical forces in the cartilage matrix23,24.
Cilia are likely involved in multiple, interlinked roles. Currently, chondrocyte cilia are known to be important in processing infor-mation about mechanical stimulation and osmotic loading25. We will next examine the evidence for the sensory function of cilia, particularly in mechanotransduction.
Fig. 1. Structural relationship between the extracellular matrix (Em), the ciliary axoneme (Ax), the distal (Dc) and proximal (Pc) centrioles, and the Golgi apparatus (G). The distal centriole acts as the basal body of the cilium. Arrow¼ transitional fiber. Bar¼ 500 nm. Reprinted with permission from Jensen et al.23.
The sensory role of cilia
Cilia in mechanotransduction
Support for the role of chondrocyte cilia in mechano-transduction includes evidence from other cell types, the require-ment of chondrocyte cilia for responses to mechanical loading, the presence of mechanotransduction proteins on the cilia, and upre-gulation of both ciliary genes and mechanotransduction genes in response to compression.
Embryologically, the primary cilium determines symmetry, and mutations in cilia proteins result in abnormalities in several tissues, including articular joints26. Cilia are also present in cells from adult tissues and adult cell types, such as kidney tubules, bile duct, neurons, pancreas, thyroid, andfibroblasts, suggesting a role for cilia beyond development27. Further, cilia in some of these cell types, including chondrocytes, are thought to be related to mechanotransduction. For instance, in renal epithelial cells, the primary cilium detectsfluid shear forces and causes the cells to respond to fluid flow with changes in intracellular Ca2þ26. An
importantfinding in understanding polycystic kidney disease was the localization of polycystins to cilia in renal epithelial cells and the mediation of Ca2þ response to mechanosensation by ciliary polycystins26. Intracellular Ca2þsignaling controls gene and protein expression in response to mechanotransduction, so Ca2þinflux or Ca2þ signaling is often used as an indicator of mechano-transduction3. In one study, renal epithelial cells from knockout
mice deficient in the ciliary protein polycystin-1 did not respond with Ca2þtofluid flow, and neither did wild-type cells treated with antibody to inactive polycystin-126. In a similar process in osteo-cytes, the mechanosensitive protein polycystin-1 on cilia is cleaved when cilia bend and triggers release of internally stored Ca2þ as opposed to a Ca2þinflux24.
Requirement of chondrocyte cilia for biochemical responses to mechanical loading
Similarly, mechanical loading activates intracellular Ca2þ in chondrocytes28. However, loading stimulates intracellular Ca2þ only in chondrocytes with cilia9. Chondrocytes isolated from transgenic mice with mutant Polaris, a necessary protein for cilia formation, failed to initiate a mechanotransduction response9. Compression in these chondrocytes also failed to stimulate aggre-can gene expression and glycosaminoglyaggre-can production9. Thus, cilia are necessary for mechanotransduction in chondrocytes. Un-like in other cell types, cilia are required for downstream events that process the mechanical signal, but not the initial mechanore-ception9. Chondrocyte cilia are oriented either toward the articular surface or toward the subchondral bone29, but some studies report that cilia point away from the subchondral bone12. The orientation toward the articular surface is more pronounced in chondrocytes of load-bearing cartilage and is inconsistent in noneload-bearing cartilage, further suggesting involvement of cilia in mechanotransduction29.
The molecules of mechanotransduction
Mechanotransduction in the chondrocyte includes integrins, Ihh, Ca2þ, ion channels, connexins, purine, and cyclic adenosine monophosphate (cAMP) second messenger signaling. The cilia serves as a compartment for many of these signaling pathways25.
Integrins pass through the cell membrane and attach the cell to the ECM, linking the cytoskeleton inside the cell to the ECM outside of the cell. Mechanical force induces conformational
changes in integrins that result in a stronger integrin-ECM connection30. Integrins
a
2,a
3, andb
1, and the proteoglycan NG2 are present on chondrocyte primary cilia and are thought to mediate contact with ECM molecules31. Because other key ECM attachment proteins are absent in cilia, but present in chondrocyte plasma membranes, it is thought that the plasma membrane ceptors are important for attachment to the ECM and cilia re-ceptors are important in sensing the pericellular environment farther away from the cell31.The increase in Ihh signaling driven by mechanical loading re-quires the presence of chondrocyte cilium25,32. The Ihh receptor Patched 1 (Ptc1) and its regulatory target Smoothened (Smo) localize to the cilia33. Smo is released when the matrix protein Ihh binds to Ptc133. Another matrix protein,fibroblast growth factor
(FGF)-2, mediated loading-induced extracellularly regulated ki-nase34. A link between FGF and cilia has been identified in other cell types, but not yet in chondrocytes35.
Transient receptor potential vanilloid 4 (TRPV4), a Ca2þchannel, mediated Ca2þsignaling induced by mechanical loading in porcine articular chondrocytes3. TRPV4 has been found on cilia in renal cells36and on chondrocyte cell membrane and chondrocyte cilia37. TRPV4 mutations disrupt skeletal development and joint degen-eration3. Acid-sensing ion channels have been shown in other cell types to be activated by mechanosensation and are present in rat articular chondrocytes38. It is unknown if these acid-sending ion channels localize to the cilia.
Connexin 43, a mechanosensitive adenosine triphosphate (ATP)-release channel, is found on chondrocyte cilia39. Like Ca2þ influx, ATP is a second messenger in many systems, including mechanotransduction. Once released from the cell, ATP binds to purine receptors. Several P2X and P2Y purine receptors have been identified on human articular chondrocytes39.
Mechanical loading increases cAMP, another common second messenger, in chondrocytes40and other cell types33. This response requires the presence of primary cilia in osteocytes in a Ca2þ -in-dependent process, and the adenylyl cyclase enzyme AC6 which catalyzes the generation of cAMP localizes to cilia in these cells33.
Upregulation of both ciliary genes and mechanotransduction genes in response to compression
In one study41, microarray analysis of differential gene expres-sion after compresexpres-sion identified genes known to be involved in mechanosensory roles, such as the ECM protein Biglycan, the ma-trix metalloproteinase MMP9, a matrix metalloproteinase-regulatory protein Cited2, and Ahnak, a protein involved in intra-cellular Ca2þsignaling. Wnt signaling molecules and other genes linked to ciliary function that were not previously known to be involved in mechanosensation were also identified41.
Cilia sense osmoticfluctuations
Cartilage compression and relaxation combined with the negatively-charged proteoglycan-rich ECM results in dramatic changes in water content and osmoticfluctuations21. OA is
char-acterized by a loss of proteoglycans and reduced interstitial os-molarity21. Using a novel live microscopy technique, Rich et al.21 established that chondrocyte cilia respond within minutes to changes in osmolarity with changes in length. Thus, cilia may have a sensory role for multiple stimuli.
IFT in chondrocyte cilia
Beyond mediating mechanotransduction, cilia also respond to compression. In a study of chondrocyte/agarose constructs, cyclic
compression reduced the number and length of cilia42. In another study, bovine OA chondrocytes had more and longer cilia than chondrocytes from healthy cartilage16. In kidney epithelium, cilia length is proportional to the magnitude of Ca2þinflux initiated by cilial deflection43.
Changes in cilia length and number may be an adaptive response to repeated or prolonged mechanical stimulation, as has been reported for algalflagella44. Chondrocyte cilia length changes
in response to the inflammatory cytokine interleukin-
b
, which is highly expressed in OA, and this elongation drives chemokine release, suggesting that changes in cilia length are involved in inflammation45. Changes in the number and length of cilia may bean adaptive response to mitigate the adverse effects of mechanical loading in an unstable joint, a deleterious response that exacerbates OA, or a symptomatic phenotype with only diagnostic significance. While the significance of such changes is not well understood, a mechanism can be postulated involving IFT and Hedgehog (Hh) signaling.
IFT is responsible for assembly, disassembly, and maintenance of cilia andflagella and may be regulated by kinases, Hh, and Wnt pathways11,42. Hh, Wnt, and IFT proteins link cilia formation and chondrocyte differentiation11. The details of Hh signaling in the primary cilium are elegant and well characterized. In the unbound state, Ptc1 represses Smo. When Ihh binds Ptc1, Smo is released, translocates to the tip of the primary cilium, and activates Gli transcription factors24,46,47. Impairment of IFT disrupts Hh signaling25. Mechanical strain induced Ihh expression and
activa-tion of the Hh pathway25. At high magnitude strain, cilia disas-sembly, a function of IFT, occurred and prevented Hh activation25.
Cilia and OA
Transmission electron microscopy has detected cilia in chon-drocytes within the cartilage of OA patients15,48. Links between cilia defects and abnormal cartilage have been found through studies of BardeteBiedl Syndrome (BBS), a disorder resulting from defects in ciliary proteins. Transgenic mice with knock-out mutations in BBS proteins or in the ciliary protein Polaris have cartilage abnormalities, which include an OA appearance49,50. Results from these studies suggest that BBS proteins could be targeted in future experiments to elucidate the role of cilia in mechanotransduction.
Interestingly, cilia are found on chondrogenic progenitor cells that have the potential to regenerate cartilage, suggesting a ther-apeutic target for OA through cartilage repair and regeneration27. Cilia disassembly may protect the chondrocyte from high me-chanical loading25. Intriguingly, Hh signaling was elevated in OA cartilage, and inhibiting Hh signaling reduced OA severity51. Thus, there is therapeutic potential in targeting the primary cilium, IFT, and Hh for the resolution of OA. Furthermore, therapeutic interest in the role of cilia trafficking to modulate of inflammatory signaling stems from the observation that the cilium sequesters hypoxia-inducible factor 2
a
during inflammation52.Conclusions
The role of the non-motile primary cilium has been elusive for decades. The role of cilia in mechanotransduction in other cell types, the requirement of chondrocyte cilia for biochemical re-sponses to mechanical loading, the presence of proteins known to be involved in mechanotransduction on the cilia, and upregulation of both ciliary genes and mechanotransduction genes in response to compression suggest that the chondrocyte primary cilium is important for mechanotransduction. Chondrocytes are mechano-sensitive and require regular mechanical stimulation for optimal
functioning. When mechanical stimulation occurs, the primary cilium, described as a protruding switch toggled by compression24, is a vital link in the transmission of mechanical stimulation to the cell, which drives a cellular response while responding itself to mechanical stimulation through IFT.
The altered morphology of primary cilia in chondrocytes from OA cartilage suggests a role in that disease. Thus, this organelle and the signaling pathways it houses could be important targets in the prevention and treatment of OA through molecular regulation or through controlled mechanical stimulation delivered by physical therapy and light exercise.
Author contributions
Dr Ruhlen wrote the initial draft and both Dr Ruhlen and Dr Marberry edited it.
Financial disclosures
The authors have nofinancial disclosures or conflicts of interest. Conflict of interest statement
There are no competing interests.
Acknowledgments
We appreciate the technical assistance of Deborah Goggin, MA.
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