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The solitary (primary) cilium–A mechanosensory toggle switch in bone and cartilage cells
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Cellular Signalling 20 (2008) 1019 – 1024 www.elsevier.com/locate/cellsig
Review
The solitary (primary) cilium–A mechanosensory toggle switch in bone and cartilage cells J.F. Whitfield ⁎ Institute for Biological Sciences, National Research Council of Canada, Building M-54, Montreal Road Campus, Ottawa, Ontario, Canada K1A 0R6 Received 11 November 2007; received in revised form 3 December 2007; accepted 3 December 2007 Available online 8 December 2007
Abstract Osteocytes and articular chondrocytes sense and respond to the strains imposed on bones and joints by various activities such as breathing and walking. This mechanoresponsiveness is needed to maintain bone and cartilage microstructure and strength. In bone the large number of osteocytes form a vast osteointernet in which the gap junctionally interconnected members are lodged in an extensive lacunocanalicular network. The much smaller number of articular chondrocytes are not interconnected in a chondrointernet. Instead, they are separately lodged in capsules called chondrons. While there are many possible strain-sensing devices, it now appears that the non-motile solitary (primary) cilia protruding like aerials from osteocytes (as well as osteoblasts) and chondrocytes are switches that when toggled by cyclical pulses of lacunocanalicular fluid or cartilage compression send signals such as Ca2+ surges into the cell to trigger a cascade of events that include appropriate gene activations to maintain and strengthen bone and cartilage. Moreover, the chondrocyte cilium with its Ihh(Indian hedgehog)-activated Smo receptor is a key player along with PTHrP in endochondral bone formation. © 2007 Elsevier Inc. All rights reserved. Keywords: Articular cartilage; Ca2+; Chondrocytes; Chondron; Endochondral bone formation; Ihh (Indian hedgehog); IP3; solitary (primary) cilium; Kidney cells; Lacunocanalicular network; Osteoblasts; Osteocytes; PKD1 (PC-1); Ptc (patched); PTHrP (parathyroid hormone-like peptide); Smo (smoothened); TRPP2 (PC-2)
Contents 1. Introduction: the osteocytic osteointernet . . . . . . . . . . . . . 2. Cyclical bone strain and ‘sloshing’ lacunocanalicular fluids . . . . 3. The solitary (primary) cilium, an osteocyte’s mechanoresponsive 4. The chondrocyte cilium. . . . . . . . . . . . . . . . . . . . . . 5. Conclusion\cilia and the lively skeleton. . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction: the osteocytic osteointernet Superficially bones are inert objects that simply serve as levers for muscles and store-rooms for 99% of the body’s calcium. As ⁎ Tel.: +1 613 722 2209. E-mail address: [email protected]. 0898-6568/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2007.12.001
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anyone who has broken open a bone knows, they consist of a hard shell, the cortex, that encloses a network of struts and plates. The cortex is made of a hard, 5–10% porous, hydroxyapatite-containing matrix with an embedded network of interconnected osteocytes that are fed and regulated by blood vessels and nerves running through a system of major pipelines known as Haversian and Volkmann’s canals [1,2]. The cortex provides the attachment
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sites for muscles and tendons. By contrast, the internal cancellous or trabecular bone is 75%–90% porous with cavities containing hematopoietic red marrow or fatty yellow marrow. The struts and plates of the trabecular lattice are aligned with the bone’s stress forces which enables the bone to work efficiently with minimum strain and minimum mass. The trabecular lattice provides an immense surface for the interaction of bone cells with various hormones and cytokines to release Ca2+ into the blood when needed. Moreover the trabeculae also provide niches (nests) carpeted with retired osteoblasts, called bone-lining cells, that anchor and regulate the long-term hematopoietic stem cells [3]. Despite their outward appearance, bones are far from simple levers, stem cell nests and Ca2+ storehouses. On the contrary, they are equipped to respond to the strains inflicted on them by muscle pulling and feet striking the ground while walking and running [1]. Surprisingly, they know where they are and the expected loading ranges of the bones they are living in, such as the very lightly strained cranial bones or heavily strained femurs, ribs, tibias, and ulnas (reviewed in [2]). Depending on its position in the skeleton a bone needs a certain amount of loading to maintain its normal structure. Without adequate loading (e.g., in the microgravity of a space ship or in immobile limbs of a paraplegic or bedridden patient) bones weaken and may fracture when reloaded. But even the strains from normal activities can produce wear and tear in the form of microcracks at ‘hot’ stress points in bones. These are promptly dug out by osteoclasts and filled with new bone by osteoblasts, a process known as remodeling [1,2]. Obviously bones have sensors that can monitor loading for maintenance and switch on repair processes when needed [2,4]. Thus we come to the bones’ ‘professional’ strain sensors, the osteocytes [1,2,4]. They are the most numerous bone cells. Human cortical bone contains about 14,000 of them per mm3 [1]. Osteocytes are retired osteoblasts that have retooled themselves from bone-making to strain-sensing [1,2]. They are the lucky members (about 4%) of osteoblast bone-making crews who avoided apoptotic down-sizing upon completion of the job. They escaped this fate because they were trapped in the bone matrix they were making. This entrapment induced them to make MT1MMP metalloproteinase which converted latent matrix TGF-β into active TGF-β which generated the signal that prevented apoptogenesis (reviewed in [2]). Locked into tiny cells called lacunae, they irreversibly switched off their bone-making genes, shrank down from plump osteoblasthood and extended 40–60 processes (‘dendrites’), each of which tunneled through the new matrix with its MT1-MMP protease to make tiny chambers called canaliculi (reviewed in [2]).The tunneling processes connected with gap junctions to other osteocytes’ processes and to the blood supply via the flat, reversibly retired osteoblasts lining the walls of the local Haversian and Volkmann canals. These so-called bonelining cells can re-retool themselves into plump functioning osteoblasts if signaled to do so by blood-borne factors such as injected PTH (parathyroid hormone) or repair-triggering products from the connected osteocytes (reviewed in [2]). Thus, osteocytes are plugged into a massive 3-D syncytium called the CCN (connected cellular network) by Sharma et al. [4] or the osteointernet by this author [2]. The extent of the osteointernet is indicated by the 500,000 to 1.2 million osteocyte processes coursing through
the canaliculi linking to each other in a single mm3 of human cortical bone. This means that there is an immense amount of ‘blogging’ going on in bones mainly about ongoing stresses, strains and microcracks. 2. Cyclical bone strain and ‘sloshing’ lacunocanalicular fluids While the osteocytes’ fluid-filled L-C (lacunocanalicular) network occupies only about 1% of a bone’s volume, the total surface area of the osteointernet’s lacunae and canaliculi radiating out from them in an adult human skeleton is about 1200 m2 compared to the 3.2 and 9 m2 of the Haversian and Volkmann canals respectively [1,5]. Thus bone is really a stiff, but compressible, fluid-filled calcareous sponge. The amplitude and frequency of pulses of L-C fluid coursing through a bone’s cortical osteointernet is determined for example by how strongly and how frequently muscles pull on the ribs during breathing cycles and the force and frequency of muscle pulling and ground strikes reverberating through the lower limb bones and hips during walking and running cycles. When isolated bone cells are shear-strained by pulsing fluid they respond with intracellular Ca2+ surges, COX (cycloöxygenase)-2 expression and PGE2 (prostaglandin E2) as well as NO production [4,6–11]. Osteocytes are much more sensitive to fluid flow, than osteoblasts (4, 9–11). And they are more readily stimulated by the cyclic strain from a train of fluid pulses rather than the static strain from a continuous compression or a constant fluid flow\the cells’ mechanosensor must be toggled on and off [4]. For example, according to Klein-Nulend et al. [11] osteocytes respond much more strongly to a pulsatile fluid flow than to intermittent thousands-fold greater hydrostatic compression forces. The cyclic loading of a bone such as a femur or tibia during walking produces pulsing fluid shear forces in the L-C network [4]. Besides subjecting the osteocytes to shear forces, the loadinginduced compression pumps waste out of the network and into the local Haversian and Volkmann canals while the subsequent relaxation sucks oxygenated and nutrient-loaded fluid from the blood back through the L-C network. When the canalicular network is severed by a microcrack or the skeletal activity falls below a critical level the osteocytes will starve and die and the susceptibility of the bone to fracture will rise. Thus, the strong cyclical pulling of muscles on hip, femurs and tibias plus the bombardment of the bones by blows from feet on the ground during walking or running or the cyclical muscle pulling on ribs during breathing cause fluid to surge back and forth through the bones’ L-C networks within certain frequency ranges that keep the osteocytes optimally oxygenated and nourished and sending bone strength-maintaining signals through the osteointernet. Obviously osteocytes must have a flow-metering device that is switched on and off by the pulsing L-C fluid and triggers Ca2+ surges, the expression of the COX-2 gene for PGE2 production and induces the expression of NO synthase that makes NO [4,6]. Sharma et al. [4] have summarized several possible pulseresponding devices. Among these are stretch-activated ion channels and/or integrin-attached signal wires that tether the osteocyte processes to the canalicular walls. When fluid pulses
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travel along the canaliculi they pry open the ion channel, tug the integrin-linked signal wires and deform the cell membrane, all of which trigger various signals, deform the cell membranes and attached cytoskeletal actin filaments and release gene-controlling protein complexes (‘mechanosomes’) from membrane scaffolds. To quote Janmey and McCulloch [12] “…..mechanotransduction, the process by which cells convert mechanical stimuli into biochemical responses, may not be a finite single process but may instead be a series of interrelated processes that involve the recruitment of a wide variety of cell attachment, cytoskeletal, and signaling proteins”. But there is a device, which until now has been unnoticed or ignored for more than three decades, that could be the osteocyte’s major mechanosensor as well as the chondrocyte mechanosensor to be discussed further on. This device is the solitary (primary) cilium [13]. 3. The solitary (primary) cilium, an osteocyte’s mechanoresponsive toggle switch “The Primary Cilium: What once did nothing, now does everything”…C.T. Anderson and T. Stearns [37th International Sun Valley Workshop on Skeletal Tissue Biology, August 5th–8th, 2007]. In 1898, the Swiss anatomist, K. Zimmermann, was the first to describe the solitary (primary) cilium protruding from the cells of a surprisingly wide variety of tissues [14]. Of course since it was obviously not needed to move the immobile cells in these tissues, it was dismissed as being just a functionless relict of early cell evolution\a sort of cellular appendix. Zimmermann had not looked at bones. This omission was corrected in 1972 by Tonna and Lampen [15]. But no attention was paid to the osteoblast/osteocyte cilium from 1972 to 2003 when I [13] proposed that the solitary cilium they saw could be a mechanosensing switch toggled in bones by pulsing L-C fluid. This was prompted by the discovery by Praetorius and Spring [16] and Nauli et al. [17] that the single, stiff, 8-μm solitary cilium protruding like a micro-aerial from each canine and murine embryo kidney tubule cell is a flowmeter. The solitary cilium grows from the mature mother centriole of the mother–daughter pair of centrioles in a cell’s centrosome located between the nucleus and the Golgi apparatus [18]. This cilium is not motile. Motile cilia have 9 peripheral microtubule doublets connected to 2 central singlet microtubules and the motors to drive them, but the solitary cilium only has the 9 peripheral doublets without the pair of central motor-attached microtubules [18]. Although it is not motile, the solitary cilium is a very busy device with kinesin and dynein motors carrying tubulin, receptors, stretch-activated ion channel components and other cargos back and forth respectively from the cilium’s basal body. It now appears that its jobs in various tissues are: to ‘sniff’ the external fluid with various receptors such as kidney cell EGF receptors, neuronal 5-HT6 serotonin receptors and SST3 somatostatin receptors, fibroblasts’ FGF receptor, serumstarved NIH3 T3 murine fibroblasts’ PDGFαα receptors, and hedgehog’s Smo•Ptch (smoothened-patched) receptors; to measure fluid currents by bending and opening its stretch-
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opened ion channels; and to stimulate gene expressions by the stretch-induced proteolysis and release of transcription factors from certain membrane proteins [18–22]. How could bending cilia send signals between osteocytes in the osteointernet? The original model for this would be the kidney cilium which has in its membrane the Gi/o-protein-coupled mechanosensor, PKD1 (polycystic kidney disease-1) or PC-1 (polycystin-1) with 11 TMDs (transmembrane domains) and its 200–226 residue C-tail tied to the C-tail of TRPP2 (transient receptor potential protein 2) also known as PC-2 (polycystin-2) a 6-TMD strain-configurable non-specific Ca2+-permeable cation channel [20, 23–28]. Since the heteromeric PKD1(PC-1)•TRPP2 (PC-2) complex can respond to mechanical strain by activating its Gi/o-protein at the same time morphing into a cation channel, Delmas [20] has called it a ‘signalplex’. Bending the kidney cell cilium with a micropipette or by increasing the fluid flow rate from 2 to 8 μl/s stretches PKD1 (PC-1) which in turn pulls open TRPP2 (PC-2) to let Ca2+ surge into the cilium and trigger a cascade of events that includes PLC activation and the release of IP3 (1, 3, 5-inositol trisphosphate) from the cilium membrane’s phosphoinositides [16,17,23,26,29–33] (Fig. 1). Since Ca2+ does not pass through gap junctions (it closes them), the IP3 takes over and starts a wave of Ca2+ surges spreading from the cell with the toggled cilium to connected neighboring cells by passing through the intercellular gap junctions and releasing Ca2+ from the neighbors’ internal stores. If osteoblasts and osteocytes have toggle switches that operate like the kidney tubule cells’ switch, they should also have PKD1 (PC-1)• TRPP2 (PC-2) signalplexes in their solitary cilia. Indeed, Xiao et al. [34] have reported that murine MC3T3 preosteoblasts and MLO-Y4 osteocytes express the Tg737 and Kif3a genes for cilial proteins, express the genes for the PKD1 (PC-1) and TRPP2 (PC-2) signalplex. proteins and indeed have the solitary (primary) cilium protruding from them. This means that they should have ciliary signalplexes and should respond to fluid flow with external Ca2+ surging into the cell through TRPP2 (PC-2) ion channels. In fact they need PKD1 (PC-1) to activate the P1 promoter of the gene for the Runx2 transcription factor which targets the osteoblast’s genes for α1 (I) procollagen, osteocalcin, osteopontin, and osterix [34] (Fig. 1). Thus, an overactive PKD1(PC-1) causes Runx2 overexpression and increased osteoblast differentiation and osteogenesis. By contrast, mutant Pdk1−/− mice lacking PKD1 (PC-1) have reduced trabecular bone volume and density, reduced cortical bone thickness, and decreased bone mineral apposition rate. In other words, without PKD1 (PC-1), bone formation is impaired and the mutant mice are osteopenic. Thus, osteoblasts do appear to have a cilium switch which when toggled by L-C fluid pulses drives bone maintenance or formation (Fig. 1). Moreover Malone et al. [35] have shown that toggling the MC3T3 cell’s cilium with fluid flow also raises the osteoclastsuppressing OPG (osteoprotegerin)/RANKL (osteoclasts stimulator) expression ratio which means that cilium toggling L-C fluid pulses can also keep bones from being resorbed by osteoclasts [2]. My 2003 proposal [13] was based on the kidney’s cilium signaling model according to which L-C fluid pulsing starts the signaling process with the formation of TRPP2 (PC-2) channels and external Ca2+ flowing through them into the cilium and the
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(PC-2) channels? Indeed bending a cilium can cause PKD1 (PC-1) to activate its Gi/o and stimulate the intramembrane proteolysis of the PKD1 (PC-1) protein with the release of its C-terminal tail [19,20,25]. The released C-terminal tail can then travel to the nucleus and activate several genes but most importantly the master osteogenes’ activator Runx2 (Fig. 1). Furthermore, PKD1 (PC-1) has a Gi/o-protein-activating domain the bending-induced activation of which might stimulate a phospholipase and the production of the internal Ca2+-releasing IP3 (Fig. 1). But since Xiao et al. [34] found that overexpressing various constructs of the PKD-1 (PC-1) C-tail without the G-protein-coupling region but with the TRPP-2 (PC-2)-coupling coiled coil region could restore Runx2 expression in the mutant mice, the osteoblast cilium may indeed have the PKD1(PC-1)•TRPP2 (PC-2) signalplex. Obviously a lot more work must be done to identify the osteoblast cilium’s signaling gadget and the signals it sends into the cell when its cilium is toggled by L-C fluid pulses (Fig. 1). 4. The chondrocyte cilium
Fig. 1. Cyclical straining of bone during various activities such as breathing or walking, causes pulses of fluid coursing through the L-C network. These pulses toggle the cilium protruding like an aerial from the network’s mechanosensing osteocytes. According to the available information [34,35], the bending of the cilium stretches and thus induces the cleavage of the mechanosensitive PKD1 on the cilium membrane. This results in the translocation of the PKD1 C-terminal tail (CTT) to the nucleus [19,25] which, reasoning from the work of Xiao et al. [34], targets the gene for the Runx2 transcription factor which in turns targets key osteogenesis-driver genes such as α1(I) procollagen, osteocalcin, osteopontin, and osterix [34]. Disabling the PKD1 gene, impairs bone formation and maintenance and causes osteopenia [34]. In kidney cells PKD1 (PC-1) is linked to TRPP2 (PC-2) which morphs into a Ca2+-permeable ion channel when PKD1 is stretched. Although osteocytes have TRPP2 (PC-2) [34], the Ca2+ surge triggered by toggling the cilium is not from external Ca2+ flowing into the cell through opened channels, but from internal stores [35]. Indeed the stretchactivated PKD1 by itself could have caused the release of internally stored Ca2+ [20]. Therefore the uncertainty of TRPP2 function in the strained osteocyte is indicated by a broken line and a question mark.
cell (Fig. 1). At first this seemed reasonable because the fluid pulses that Malone et al. [35] have directly demonstrated to bend solitary cilia back and forth has been known since ‘pre-cilium times’ to cause Ca2+ oscillations in murine and rat osteoblastic cells and to stimulate c-fos and COX-2 gene expressions [6– 8,36]. But there is a problem with extending the kidney cilium model to bone cells. Chen et al. [6] had already shown that the Ca2 + that stimulated c-fos/COX-2 expression in their experiments did not come into the cell through channels but from internal stores . Now Malone et al. [35] have shown that bending MC3T3 preosteoblast and MLO-Y4 osteocyte cilia with fluid pulses does stimulate COX-2 gene expression and PGE2 production. Moreover, they have also shown that toggling the bone cells’ cilia does not cause intracellular Ca2+ surges by opening channels which it should if they had complete PKD1(PC-1)•TRPP2(PC-2) signalplexes (Fig. 1). Does this mean that the Ca2+ and Runx2-driven osteogenic gene responses to the bending of bone cell cilia is due entirely to the stretching/activation of PDK1 (PC-1) without opening TRPP2
As you are walking and breathing with your L-C fluids pulsing through osteointernets and toggling osteocyte cilia, the compressive forces on the cartilage in your knees shot up from 1 to 2 atm (101.3–202.6 kPA) to 100–200 atm (1.01–2.03 × 104 kPA) when you stood up and are now cycling between 40 and 50 atm (4.05– 5.06× 103 kPA) as you are walking [37]. This strong cyclical vertical compression does not go unnoticed by the chondrocytes in capsules called chondrons [38]. Unlike the highly social osteocytes communicating with each other through their extensive syncytial osteointernet, the very long-lived articular chondrocytes (there are about 10,000/mm3 of femoral head cartilage) do not live in a well-fed and oxygenated, gap-junctionally interconnected syncytium with access to the blood circulation\there is no chondrointernet. They live in an avascular, hence very low-oxygen, fiber-reinforced gel where they must depend entirely on the diffusion of oxygen and nutrients through the gel and depend on glucose transporter I and the anaerobic glycolysis machinery to make their ATP [2,37]. They are thus limited in their activities by this restricted fuel supply compared to the osteocytes with their substantial mitochondrial complements and L-C contact with the blood circulation. Nevertheless, chondrocytes still sense and respond to the movements of their joints. So how might an antisocial, oxygen-deprived, articular chondrocyte, with its limited ATP making ability, feel the movement of its joint? One of the ways, maybe the most important one, is with a compression-toggled solitary (primary) cilium. The chondrocyte cilium was first mentioned by Hart in a 1968 abstract [39] and a decade later by Wilsman and colleagues [39–41]. Since then it has been studied largely by C.A. Poole and his colleagues in Auckland New Zealand long before the osteoporosis-driven explosion of interest in bone signaling and now the osteoblast/osteocyte cilium. Chondrocytes are encapsulated in fibrillar-walled chondrons which are embedded in an ICM (interchondronal matrix), a hydroelastic collagenous composite [38]. The chondron is distinguished by being selectively enriched with the minor collagen VI and water-binding proteoglycans (hyaluoronan and aggrecan)
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[38,42]. Because of the water associated with the proteoglycans, the first person to recognize the chondron, Benninghof [43], regarded them as fluid-filled sacs or bladders. In fact when the cartilage and its chondrons are strongly and cyclically compressed as their owner stands up and starts walking the water associated with the chondron proteoglycans is squeezed through the PCC (pericellular capsule) wall into the much less strained ICM (i.e., the PCC has a smaller compression modulus than the ICM; it takes less force to produce a given amount of PCC strain than ICM strain) leaving the chondron with a higher osmotic pressure because of the compression-concentrated proteoglycans and salts. Along with the water squeezed out during each compression cycle, various chondrocyte products including wastes and those needed to maintain the joint are filtered through the PCC meshwork The intrachondronal pressure is prevented by the strong PCC from dissipating and the chondron collapsing. When the compression drops during each cycle, water with chondrocyte nutrients is drawn back into the decompressed chondron because of the high osmotic pressure produced during compression [38]. By straining the PCC’s fibrillar meshwork and pushing the PCC wall closer to the cell surface during the flattening of the chondron during each compression cycle the chondrocyte cilium with meshwork fibrils attached to it will be bent [44,45] (Fig. 2); (see also the chondron cartoons in Fig. 5 of Poole [38]). The upper part of the cilium is decorated with plaques containing α2β1 and α3β1 integrins which are tethered to the pericellular collagen fibers [44,45] (Fig. 2). The bending of the cilium by an encroaching PCC wall and/or the tugging on the integrins by pericellular collagen fibrils in the flatterning chondron will generate a cascade of events ranging from internal Ca2+ release to chondrocyte-appropriate gene activations [46] (Fig. 2) We do not
Fig. 2. Unlike the osteocytes in their osteointernet, articular chondrocytes are not parts of a chondrointernet. Instead they are enclosed in chondron capsules suspended in an avascular collagenous composite. The chondrocyte cilium is decorated in its upper part with α2β1 and α3β1 integrins-containing plaques to which are attached collagen (e.g., collagen VI) meshwork fibrils. When the cartilage is compressed while standing, walking, or running the cilium is bend by the encroaching chondron wall and by being pulled by the collagen fibrils in the compressed chondron. While little is currently known about the signals sent into the chondrocyte by its bending cilium one of the signals is likely an inflow of Ca2+ and the many events this is likely to trigger.
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yet know whether the chondrocyte cilium has PKD1 (PC-1) and TRPP2 (PC-2) as do the kidney cell and osteoblast/osteocyte cilia. But according to Guilak et al. [47] and Yellowley et al. [48,49], fluid flow or deformation of the chondrocytes with a micropipette induces the inflow of Ca2+ through a mechanosensitive channel as well as the IP3-induced release of Ca2+ from internal stores. This hints at the cilia possibly having the full, toggle-activatable PKD1 (PC-1)•TRPP2 (PC-2) signalplex. While this discussion has focused on the response of the solitary (primary) cilium of an articular chondrocyte in its chondron to the strains of cyclical compression, the chondrocyte cilium has a major role in endochondral bone formation, Factors and events in the endochondral growth and development of a long bone such as the femur have been recently reviewed by Kronenberg [50] and very recently by Whitfield [2]. Endochondral bone formation is driven from cartilaginous growth plates at the ends of the bone. The round, moderately sized chondrocytes in the reserve or resting zone at the top of the growth plate nearest the epiphyses are the chondroprogenitor cells. When a progenitor cell is signaled to start a cell cycle and divides, one of its daughters stays in the home niche while the other joins one of several parallel columns or stacks of flat, proliferating, transit-amplifying cells making aggrecan, collagen II, link protein and the gene for CMP (cartilage matrix protein), the specific marker for cells in the pre-hypertrophic zone. Eventually they stop proliferating, turn off their protective anti-apoptosis Bcl-2 protein, and switch on the terminal hypertrophic program which ends in their apoptotic death and the conversion of the cartilage to vascularized bone. Cilium generation in murine growth plate chondrocytes can be prevented by disabling the gene for the Kif3a subunit of the kinesin motor that carries the various structural and functional components up along the ciliary microtubules [51]. This lack of cilia causes dwarfism by causing the misalignment of mitotic prehypertrophic chondrocytes and their daughters along the columns’ longitudinal axes and the severe shortening of the prehypertrophic zone by accelerating the onset of the terminal hypertrophic program [51]. The reason for this striking shortening is due to the absence of Smo (smoothened)•Ptc (patched) complexes that would normally have been located on the solitary cilia [21,52]. When the transitamplifying pre-hypertrophic cells stop proliferating and start terminally hypertrophying, they also start making Ihh which reaches the tops of the pre-hypertrophic columns, binds to the cells’ Ptc receptors which release active Smo from the ciliary Smo•Ptc complexes. The liberated Smo then induces the expression of TGFβ via the Gli transcription factor. The TGF-β in turn stimulates the pre-hypertrophic cells to express and release PTHrP (parathyroid hormone-related peptide) (reviewed in [2,50]). The secreted PTHrP keeps the prehypertrophic cells proliferating and prevents them from initiating the hypertrophic program and the cartilage-bone transition. Thus, the chondrocyte cilium is a major part of the feedback mechanism that determines the structure and size of the pre-hypertrophic zone and thus the length of the adult bone. 5. Conclusion\cilia and the lively skeleton According to the story so far, the solitary (primary) cilium, once believed to be just a relict of early cellular evolution,
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controls, among many other things, endochondral bone formation and the responses of mature bone and cartilage to mechanical strain. The Ihh receptor complex Smo•Ptc on the cilia of transitamplying chondrocytes in developing bone is part of the IhhPTHrP feedback mechanism that determines the length of the prehypertrophic zone and thus the final adult bone length. In adult bones, the cyclical compression produced by various activities sends pulses of fluid through the bones’ extensive L-C steointernet. These pulses toggle the osteocytes’ solitary (primary) cilia which send waves of Ca2+ and Ca2+-mobilizaing IP3 signals through the L-C osteointernet. These ciliary signals keep the osteogenic genes active to maintain an optimal bone mass and strength. Without them bones weaken and become increasingly likely to fracture if re-loaded. Unlike the extensively interconnected osteoblasts, the chondrocytes in the articular cartilage are solitary and individually enclosed in chondron capsules. The cyclic compression of the articular cartilage in the moving skeleton, flattens the chondrons with their enclosed chondrocytes. This bends the cilia which send Ca2+ signals into the cell to keep the genes for cartilage components active to maintain and renew the cartilage. While the solitary (primary) cilium now appears to be a major mechanosensory device in bone and cartilage we still know very little about how it is toggled, the signals it sends and how it relates to the other mechanosensing mechanisms. References [1] R.B. Martin, D.B. Burr, N.A. Sharkey, Skeletal Tissue Mechanics, Springer-Verlag, New York, 1998. [2] J.F. Whitfield, Growing Bone, 2ed.Landes Bioscience, Austin, TX, 2007. [3] J.F. Whitfield, Cancer Lett. 244 (2006) 8. [4] U. Sharma, A.G. Mikos S.C. Cowin, in Mechanosensory Mechanisms in Bone, R.Lanza, R. Langer, J. Vancanti eds., Chapter 61, Elsevier/ Academic Press, San Diego, 2007. [5] L.C. Johnson, Birth Defects Orig. Artic. Ser. 2 (1966) 66. [6] N.X. Chen, K.D. Ryder, F.M. Pavalko, C.H. Turner, D.B. Burr, J. Qiu, R.L. Duncan, Am. J. Physiol., Cell Physiol. 278 (2000) C989. [7] S.W. Donahue, H.J. Donahue, C.R. Jacobs, Am. J. Physiol., Cell Physiol. 281 (2001) C1635. [8] S.W. Donahue, H.J. Donahue, C.R. Jacobs, J. Biomech. 36 (2003) 35. [9] J. Klein-Nulend, P.J. Nijweide, Curr. Osteoporosis Rep. 1 (2003) 5. [10] J. Klein-Nulend, C.M. Semeins, N.E. Ajubi, P.J. Nijweide, E.H. Burger, Biochem. Biophys. Res. Commun. 217 (1995) 640. [11] J. Klein-Nulend, A. van der Plas, C.M. Semeins, N.E. Ajubi, J.A. Frangos, P.J. Neiweide, E.H. Burger, FASEB J 9 (1995) 441. [12] P.A. Janmey, C.A. McCulloch, Annu. Rev. Biomed. Eng. 9 (2007) 1. [13] J.F. Whitfield, J. Cell. Biochem. 89 (2003) 233. [14] K.W. Zimmermann, Arch. Mikrosk. Entwickl.Mech. 52 (1898) 552. [15] E.A. Tonna, N.M. Lampen, J. Gerontol. 27 (1972) 316. [16] H.A. Praetorius, K.R. Spring, J. Membrane Biol. 184 (2001) 71. [17] S.M. Nauli, F.J. Alenghat, Y. Luo, E. Williams, P. Vassilev, X. Li, A.E. Elia, W. Lu, E.M. Brown, S.J. Quinn, D.E. Ingber, J. Zhou, Nat. Genet. 33 (2003) 129. [18] P. Satir, S.V. Christensen, Annu. Rev. Physiol. 69 (2007) 377. [19] V. Chauvet, X. Tian, H. Husson, D.H. Grimm, T. Wang, T. Hieseberger, P. Igarashi, A.M. Bennett, O. Ibraghimov-Beskrovnaya, S. Somlo, M.J. Caplan, J. Clin. Invest. 114 (2004) 1433.
[20] P. Delmas, Biol. Res. 37 (2004) 681. [21] J.T. Eggenschwiler, K.V. Anderson, Annu. Rev. Cell Dev. Biol. 23 (2007) 345. [22] R. Ma, W.-P. Li, D. Rundle, J. Kong, H.I. Akbarali, L. Tsiokas, Mol. Cell. Biol. 25 (2005) 8285. [23] J.R. Davenport, B.K. Yoder, Am. J. Physiol. Renal Physiol. 289 (2005). [24] J.R. Forman, S. Qamar, E. Paci, R.N. Sanford, J. Clarke, J. Mol. Biol. 349 (2005) 861. [25] L.M. Guay-Woodford, J. Clin. Invest. 114 (2001) 1404. [26] V. Singla, J.F. Reiter, Science 313 (2006) 629. [27] C. Stayner, J. Zhou, Trends Pharmacol. Sci. 22 (2001) 543. [28] R. Witzgall, Naunyn-Schmiedeberg’s Arch. Pharmacol. 371 (2005) 342–347. [29] S.M. Nauli, J. Zhou, BioEssays 26 (2004) 844. [30] H.A. Praetorius, K.R. Spring, Annu. Rev. Physiol. 67 (2005) 515. [31] H.A. Praetorius, K.R. Spring, J. Membrane Biol. 191 (2003) 69. [32] H.A. Praetorius, K.R. Spring, Curr. Opin. Nephrol. Hypertens. 12 (2003) 517. [33] M.K. Raychowdhury, M. McLaughlin, A.J. Ramos, N. Montalbetti, R. Bouley, D.A. Ausiello, H.F. Cantiello, J. Biol. Chem. 280 (2005) 34718. [34] Z. Xiao, S. Zhang, J. Mahlios, G. Zhou, B.S. Magenheimer, D. Guo, S.L. Dallas, R. Maser, J.P. Calvet, L. Bonewald, L.D. Quarles, J. Biol. Chem. 281 (2006) 30884. [35] A.M.D. Malone, C.T. Anderson, P. Tummala, R.Y. Kwon, T.R. Johnston, T. Stearns, C.R. Jacobs, Proc. Natl. Acad. Sci. 104 (2007) 13325. [36] M. Takaoki, N. Mutakami, J. Gyotoku, Biol. Sci. Space 18 (2005) 181. [37] H. Muir, BioEssays 17 (1995) 1039. [38] C.A. Poole, J. Anat. 191 (1997) 1. [39] N.J. Wilsman, J. Ultrastruct. Res. 64 (1978) 270. [40] N.J. Wilsman, Anat. Rec. 190 (1978) 871. [41] N.J. Wilsman, C.E. Farnum, D.K. Reed-Aksamit, Anat. Rec. 197 (1980) 355. [42] F. Guilak, L.G. Alexopoulos, M.L. Upton, I. Youn, J.B. Choi, L. Cao, L.A. Setton, M.A. Haider, Ann. N.Y. Acad. Sci. 1068 (2006) 498. [43] A. Benninghof, Zeitschrft. Zellforsch. 2 (1925) 783. [44] C.G. Jensen, C.A. Poole, S.R. McGlashan, M. Marko, Z.I. Issa, K.V. Vujcich, S.S. Bowser, Cell Biol. Internat. 28 (2004) 101. [45] S.R. McGlashan, C.G. Jensen, C.A. Poole, J. Histochem. Cytochem. 54 (2006) 1005–1014. [46] J.A. Carson, L. Wei, J. Appl. Physiol. 88 (2000) 337. [47] F. Guilak, R.A. Zell, G.R. Erickson, D.a. Grande, C.T. Rubin, K.J. McLeod, H.J. Donahue, J. Orthop. Res. 17 (1999) 421. [48] C.E. Yellowley, C.R. Jacobs, Z. Li, Z. Zhou, H.J. Donahue, Am. J. Physiol. 273 (1997) C30. [49] C.E. Yellowley, C.R. Jacobs, H.J. Donahue, J. Cell. Physiol. 180 (1999) 402. [50] H.M. Kronenberg, Ann. N.Y. Acad. Sci. 1068 (2006) 1. [51] B. Song, C.J. Haycraft, H. Seo, B.K. Yoder, R. Serra, Dev. Biol. 305 (2007) 202. [52] K.C. Corbit, P. Aanstad, V. Singla, A.R. Norman, D.Y. Stanier, J.F. Reiter, Nature 437 (2005) 1018.
Cellular Signalling 20 (2008) 1019 – 1024 www.elsevier.com/locate/cellsig
Review
The solitary (primary) cilium–A mechanosensory toggle switch in bone and cartilage cells J.F. Whitfield ⁎ Institute for Biological Sciences, National Research Council of Canada, Building M-54, Montreal Road Campus, Ottawa, Ontario, Canada K1A 0R6 Received 11 November 2007; received in revised form 3 December 2007; accepted 3 December 2007 Available online 8 December 2007
Abstract Osteocytes and articular chondrocytes sense and respond to the strains imposed on bones and joints by various activities such as breathing and walking. This mechanoresponsiveness is needed to maintain bone and cartilage microstructure and strength. In bone the large number of osteocytes form a vast osteointernet in which the gap junctionally interconnected members are lodged in an extensive lacunocanalicular network. The much smaller number of articular chondrocytes are not interconnected in a chondrointernet. Instead, they are separately lodged in capsules called chondrons. While there are many possible strain-sensing devices, it now appears that the non-motile solitary (primary) cilia protruding like aerials from osteocytes (as well as osteoblasts) and chondrocytes are switches that when toggled by cyclical pulses of lacunocanalicular fluid or cartilage compression send signals such as Ca2+ surges into the cell to trigger a cascade of events that include appropriate gene activations to maintain and strengthen bone and cartilage. Moreover, the chondrocyte cilium with its Ihh(Indian hedgehog)-activated Smo receptor is a key player along with PTHrP in endochondral bone formation. © 2007 Elsevier Inc. All rights reserved. Keywords: Articular cartilage; Ca2+; Chondrocytes; Chondron; Endochondral bone formation; Ihh (Indian hedgehog); IP3; solitary (primary) cilium; Kidney cells; Lacunocanalicular network; Osteoblasts; Osteocytes; PKD1 (PC-1); Ptc (patched); PTHrP (parathyroid hormone-like peptide); Smo (smoothened); TRPP2 (PC-2)
Contents 1. Introduction: the osteocytic osteointernet . . . . . . . . . . . . . 2. Cyclical bone strain and ‘sloshing’ lacunocanalicular fluids . . . . 3. The solitary (primary) cilium, an osteocyte’s mechanoresponsive 4. The chondrocyte cilium. . . . . . . . . . . . . . . . . . . . . . 5. Conclusion\cilia and the lively skeleton. . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction: the osteocytic osteointernet Superficially bones are inert objects that simply serve as levers for muscles and store-rooms for 99% of the body’s calcium. As ⁎ Tel.: +1 613 722 2209. E-mail address: [email protected]. 0898-6568/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2007.12.001
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anyone who has broken open a bone knows, they consist of a hard shell, the cortex, that encloses a network of struts and plates. The cortex is made of a hard, 5–10% porous, hydroxyapatite-containing matrix with an embedded network of interconnected osteocytes that are fed and regulated by blood vessels and nerves running through a system of major pipelines known as Haversian and Volkmann’s canals [1,2]. The cortex provides the attachment
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sites for muscles and tendons. By contrast, the internal cancellous or trabecular bone is 75%–90% porous with cavities containing hematopoietic red marrow or fatty yellow marrow. The struts and plates of the trabecular lattice are aligned with the bone’s stress forces which enables the bone to work efficiently with minimum strain and minimum mass. The trabecular lattice provides an immense surface for the interaction of bone cells with various hormones and cytokines to release Ca2+ into the blood when needed. Moreover the trabeculae also provide niches (nests) carpeted with retired osteoblasts, called bone-lining cells, that anchor and regulate the long-term hematopoietic stem cells [3]. Despite their outward appearance, bones are far from simple levers, stem cell nests and Ca2+ storehouses. On the contrary, they are equipped to respond to the strains inflicted on them by muscle pulling and feet striking the ground while walking and running [1]. Surprisingly, they know where they are and the expected loading ranges of the bones they are living in, such as the very lightly strained cranial bones or heavily strained femurs, ribs, tibias, and ulnas (reviewed in [2]). Depending on its position in the skeleton a bone needs a certain amount of loading to maintain its normal structure. Without adequate loading (e.g., in the microgravity of a space ship or in immobile limbs of a paraplegic or bedridden patient) bones weaken and may fracture when reloaded. But even the strains from normal activities can produce wear and tear in the form of microcracks at ‘hot’ stress points in bones. These are promptly dug out by osteoclasts and filled with new bone by osteoblasts, a process known as remodeling [1,2]. Obviously bones have sensors that can monitor loading for maintenance and switch on repair processes when needed [2,4]. Thus we come to the bones’ ‘professional’ strain sensors, the osteocytes [1,2,4]. They are the most numerous bone cells. Human cortical bone contains about 14,000 of them per mm3 [1]. Osteocytes are retired osteoblasts that have retooled themselves from bone-making to strain-sensing [1,2]. They are the lucky members (about 4%) of osteoblast bone-making crews who avoided apoptotic down-sizing upon completion of the job. They escaped this fate because they were trapped in the bone matrix they were making. This entrapment induced them to make MT1MMP metalloproteinase which converted latent matrix TGF-β into active TGF-β which generated the signal that prevented apoptogenesis (reviewed in [2]). Locked into tiny cells called lacunae, they irreversibly switched off their bone-making genes, shrank down from plump osteoblasthood and extended 40–60 processes (‘dendrites’), each of which tunneled through the new matrix with its MT1-MMP protease to make tiny chambers called canaliculi (reviewed in [2]).The tunneling processes connected with gap junctions to other osteocytes’ processes and to the blood supply via the flat, reversibly retired osteoblasts lining the walls of the local Haversian and Volkmann canals. These so-called bonelining cells can re-retool themselves into plump functioning osteoblasts if signaled to do so by blood-borne factors such as injected PTH (parathyroid hormone) or repair-triggering products from the connected osteocytes (reviewed in [2]). Thus, osteocytes are plugged into a massive 3-D syncytium called the CCN (connected cellular network) by Sharma et al. [4] or the osteointernet by this author [2]. The extent of the osteointernet is indicated by the 500,000 to 1.2 million osteocyte processes coursing through
the canaliculi linking to each other in a single mm3 of human cortical bone. This means that there is an immense amount of ‘blogging’ going on in bones mainly about ongoing stresses, strains and microcracks. 2. Cyclical bone strain and ‘sloshing’ lacunocanalicular fluids While the osteocytes’ fluid-filled L-C (lacunocanalicular) network occupies only about 1% of a bone’s volume, the total surface area of the osteointernet’s lacunae and canaliculi radiating out from them in an adult human skeleton is about 1200 m2 compared to the 3.2 and 9 m2 of the Haversian and Volkmann canals respectively [1,5]. Thus bone is really a stiff, but compressible, fluid-filled calcareous sponge. The amplitude and frequency of pulses of L-C fluid coursing through a bone’s cortical osteointernet is determined for example by how strongly and how frequently muscles pull on the ribs during breathing cycles and the force and frequency of muscle pulling and ground strikes reverberating through the lower limb bones and hips during walking and running cycles. When isolated bone cells are shear-strained by pulsing fluid they respond with intracellular Ca2+ surges, COX (cycloöxygenase)-2 expression and PGE2 (prostaglandin E2) as well as NO production [4,6–11]. Osteocytes are much more sensitive to fluid flow, than osteoblasts (4, 9–11). And they are more readily stimulated by the cyclic strain from a train of fluid pulses rather than the static strain from a continuous compression or a constant fluid flow\the cells’ mechanosensor must be toggled on and off [4]. For example, according to Klein-Nulend et al. [11] osteocytes respond much more strongly to a pulsatile fluid flow than to intermittent thousands-fold greater hydrostatic compression forces. The cyclic loading of a bone such as a femur or tibia during walking produces pulsing fluid shear forces in the L-C network [4]. Besides subjecting the osteocytes to shear forces, the loadinginduced compression pumps waste out of the network and into the local Haversian and Volkmann canals while the subsequent relaxation sucks oxygenated and nutrient-loaded fluid from the blood back through the L-C network. When the canalicular network is severed by a microcrack or the skeletal activity falls below a critical level the osteocytes will starve and die and the susceptibility of the bone to fracture will rise. Thus, the strong cyclical pulling of muscles on hip, femurs and tibias plus the bombardment of the bones by blows from feet on the ground during walking or running or the cyclical muscle pulling on ribs during breathing cause fluid to surge back and forth through the bones’ L-C networks within certain frequency ranges that keep the osteocytes optimally oxygenated and nourished and sending bone strength-maintaining signals through the osteointernet. Obviously osteocytes must have a flow-metering device that is switched on and off by the pulsing L-C fluid and triggers Ca2+ surges, the expression of the COX-2 gene for PGE2 production and induces the expression of NO synthase that makes NO [4,6]. Sharma et al. [4] have summarized several possible pulseresponding devices. Among these are stretch-activated ion channels and/or integrin-attached signal wires that tether the osteocyte processes to the canalicular walls. When fluid pulses
J.F. Whitfield / Cellular Signalling 20 (2008) 1019–1024
travel along the canaliculi they pry open the ion channel, tug the integrin-linked signal wires and deform the cell membrane, all of which trigger various signals, deform the cell membranes and attached cytoskeletal actin filaments and release gene-controlling protein complexes (‘mechanosomes’) from membrane scaffolds. To quote Janmey and McCulloch [12] “…..mechanotransduction, the process by which cells convert mechanical stimuli into biochemical responses, may not be a finite single process but may instead be a series of interrelated processes that involve the recruitment of a wide variety of cell attachment, cytoskeletal, and signaling proteins”. But there is a device, which until now has been unnoticed or ignored for more than three decades, that could be the osteocyte’s major mechanosensor as well as the chondrocyte mechanosensor to be discussed further on. This device is the solitary (primary) cilium [13]. 3. The solitary (primary) cilium, an osteocyte’s mechanoresponsive toggle switch “The Primary Cilium: What once did nothing, now does everything”…C.T. Anderson and T. Stearns [37th International Sun Valley Workshop on Skeletal Tissue Biology, August 5th–8th, 2007]. In 1898, the Swiss anatomist, K. Zimmermann, was the first to describe the solitary (primary) cilium protruding from the cells of a surprisingly wide variety of tissues [14]. Of course since it was obviously not needed to move the immobile cells in these tissues, it was dismissed as being just a functionless relict of early cell evolution\a sort of cellular appendix. Zimmermann had not looked at bones. This omission was corrected in 1972 by Tonna and Lampen [15]. But no attention was paid to the osteoblast/osteocyte cilium from 1972 to 2003 when I [13] proposed that the solitary cilium they saw could be a mechanosensing switch toggled in bones by pulsing L-C fluid. This was prompted by the discovery by Praetorius and Spring [16] and Nauli et al. [17] that the single, stiff, 8-μm solitary cilium protruding like a micro-aerial from each canine and murine embryo kidney tubule cell is a flowmeter. The solitary cilium grows from the mature mother centriole of the mother–daughter pair of centrioles in a cell’s centrosome located between the nucleus and the Golgi apparatus [18]. This cilium is not motile. Motile cilia have 9 peripheral microtubule doublets connected to 2 central singlet microtubules and the motors to drive them, but the solitary cilium only has the 9 peripheral doublets without the pair of central motor-attached microtubules [18]. Although it is not motile, the solitary cilium is a very busy device with kinesin and dynein motors carrying tubulin, receptors, stretch-activated ion channel components and other cargos back and forth respectively from the cilium’s basal body. It now appears that its jobs in various tissues are: to ‘sniff’ the external fluid with various receptors such as kidney cell EGF receptors, neuronal 5-HT6 serotonin receptors and SST3 somatostatin receptors, fibroblasts’ FGF receptor, serumstarved NIH3 T3 murine fibroblasts’ PDGFαα receptors, and hedgehog’s Smo•Ptch (smoothened-patched) receptors; to measure fluid currents by bending and opening its stretch-
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opened ion channels; and to stimulate gene expressions by the stretch-induced proteolysis and release of transcription factors from certain membrane proteins [18–22]. How could bending cilia send signals between osteocytes in the osteointernet? The original model for this would be the kidney cilium which has in its membrane the Gi/o-protein-coupled mechanosensor, PKD1 (polycystic kidney disease-1) or PC-1 (polycystin-1) with 11 TMDs (transmembrane domains) and its 200–226 residue C-tail tied to the C-tail of TRPP2 (transient receptor potential protein 2) also known as PC-2 (polycystin-2) a 6-TMD strain-configurable non-specific Ca2+-permeable cation channel [20, 23–28]. Since the heteromeric PKD1(PC-1)•TRPP2 (PC-2) complex can respond to mechanical strain by activating its Gi/o-protein at the same time morphing into a cation channel, Delmas [20] has called it a ‘signalplex’. Bending the kidney cell cilium with a micropipette or by increasing the fluid flow rate from 2 to 8 μl/s stretches PKD1 (PC-1) which in turn pulls open TRPP2 (PC-2) to let Ca2+ surge into the cilium and trigger a cascade of events that includes PLC activation and the release of IP3 (1, 3, 5-inositol trisphosphate) from the cilium membrane’s phosphoinositides [16,17,23,26,29–33] (Fig. 1). Since Ca2+ does not pass through gap junctions (it closes them), the IP3 takes over and starts a wave of Ca2+ surges spreading from the cell with the toggled cilium to connected neighboring cells by passing through the intercellular gap junctions and releasing Ca2+ from the neighbors’ internal stores. If osteoblasts and osteocytes have toggle switches that operate like the kidney tubule cells’ switch, they should also have PKD1 (PC-1)• TRPP2 (PC-2) signalplexes in their solitary cilia. Indeed, Xiao et al. [34] have reported that murine MC3T3 preosteoblasts and MLO-Y4 osteocytes express the Tg737 and Kif3a genes for cilial proteins, express the genes for the PKD1 (PC-1) and TRPP2 (PC-2) signalplex. proteins and indeed have the solitary (primary) cilium protruding from them. This means that they should have ciliary signalplexes and should respond to fluid flow with external Ca2+ surging into the cell through TRPP2 (PC-2) ion channels. In fact they need PKD1 (PC-1) to activate the P1 promoter of the gene for the Runx2 transcription factor which targets the osteoblast’s genes for α1 (I) procollagen, osteocalcin, osteopontin, and osterix [34] (Fig. 1). Thus, an overactive PKD1(PC-1) causes Runx2 overexpression and increased osteoblast differentiation and osteogenesis. By contrast, mutant Pdk1−/− mice lacking PKD1 (PC-1) have reduced trabecular bone volume and density, reduced cortical bone thickness, and decreased bone mineral apposition rate. In other words, without PKD1 (PC-1), bone formation is impaired and the mutant mice are osteopenic. Thus, osteoblasts do appear to have a cilium switch which when toggled by L-C fluid pulses drives bone maintenance or formation (Fig. 1). Moreover Malone et al. [35] have shown that toggling the MC3T3 cell’s cilium with fluid flow also raises the osteoclastsuppressing OPG (osteoprotegerin)/RANKL (osteoclasts stimulator) expression ratio which means that cilium toggling L-C fluid pulses can also keep bones from being resorbed by osteoclasts [2]. My 2003 proposal [13] was based on the kidney’s cilium signaling model according to which L-C fluid pulsing starts the signaling process with the formation of TRPP2 (PC-2) channels and external Ca2+ flowing through them into the cilium and the
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(PC-2) channels? Indeed bending a cilium can cause PKD1 (PC-1) to activate its Gi/o and stimulate the intramembrane proteolysis of the PKD1 (PC-1) protein with the release of its C-terminal tail [19,20,25]. The released C-terminal tail can then travel to the nucleus and activate several genes but most importantly the master osteogenes’ activator Runx2 (Fig. 1). Furthermore, PKD1 (PC-1) has a Gi/o-protein-activating domain the bending-induced activation of which might stimulate a phospholipase and the production of the internal Ca2+-releasing IP3 (Fig. 1). But since Xiao et al. [34] found that overexpressing various constructs of the PKD-1 (PC-1) C-tail without the G-protein-coupling region but with the TRPP-2 (PC-2)-coupling coiled coil region could restore Runx2 expression in the mutant mice, the osteoblast cilium may indeed have the PKD1(PC-1)•TRPP2 (PC-2) signalplex. Obviously a lot more work must be done to identify the osteoblast cilium’s signaling gadget and the signals it sends into the cell when its cilium is toggled by L-C fluid pulses (Fig. 1). 4. The chondrocyte cilium
Fig. 1. Cyclical straining of bone during various activities such as breathing or walking, causes pulses of fluid coursing through the L-C network. These pulses toggle the cilium protruding like an aerial from the network’s mechanosensing osteocytes. According to the available information [34,35], the bending of the cilium stretches and thus induces the cleavage of the mechanosensitive PKD1 on the cilium membrane. This results in the translocation of the PKD1 C-terminal tail (CTT) to the nucleus [19,25] which, reasoning from the work of Xiao et al. [34], targets the gene for the Runx2 transcription factor which in turns targets key osteogenesis-driver genes such as α1(I) procollagen, osteocalcin, osteopontin, and osterix [34]. Disabling the PKD1 gene, impairs bone formation and maintenance and causes osteopenia [34]. In kidney cells PKD1 (PC-1) is linked to TRPP2 (PC-2) which morphs into a Ca2+-permeable ion channel when PKD1 is stretched. Although osteocytes have TRPP2 (PC-2) [34], the Ca2+ surge triggered by toggling the cilium is not from external Ca2+ flowing into the cell through opened channels, but from internal stores [35]. Indeed the stretchactivated PKD1 by itself could have caused the release of internally stored Ca2+ [20]. Therefore the uncertainty of TRPP2 function in the strained osteocyte is indicated by a broken line and a question mark.
cell (Fig. 1). At first this seemed reasonable because the fluid pulses that Malone et al. [35] have directly demonstrated to bend solitary cilia back and forth has been known since ‘pre-cilium times’ to cause Ca2+ oscillations in murine and rat osteoblastic cells and to stimulate c-fos and COX-2 gene expressions [6– 8,36]. But there is a problem with extending the kidney cilium model to bone cells. Chen et al. [6] had already shown that the Ca2 + that stimulated c-fos/COX-2 expression in their experiments did not come into the cell through channels but from internal stores . Now Malone et al. [35] have shown that bending MC3T3 preosteoblast and MLO-Y4 osteocyte cilia with fluid pulses does stimulate COX-2 gene expression and PGE2 production. Moreover, they have also shown that toggling the bone cells’ cilia does not cause intracellular Ca2+ surges by opening channels which it should if they had complete PKD1(PC-1)•TRPP2(PC-2) signalplexes (Fig. 1). Does this mean that the Ca2+ and Runx2-driven osteogenic gene responses to the bending of bone cell cilia is due entirely to the stretching/activation of PDK1 (PC-1) without opening TRPP2
As you are walking and breathing with your L-C fluids pulsing through osteointernets and toggling osteocyte cilia, the compressive forces on the cartilage in your knees shot up from 1 to 2 atm (101.3–202.6 kPA) to 100–200 atm (1.01–2.03 × 104 kPA) when you stood up and are now cycling between 40 and 50 atm (4.05– 5.06× 103 kPA) as you are walking [37]. This strong cyclical vertical compression does not go unnoticed by the chondrocytes in capsules called chondrons [38]. Unlike the highly social osteocytes communicating with each other through their extensive syncytial osteointernet, the very long-lived articular chondrocytes (there are about 10,000/mm3 of femoral head cartilage) do not live in a well-fed and oxygenated, gap-junctionally interconnected syncytium with access to the blood circulation\there is no chondrointernet. They live in an avascular, hence very low-oxygen, fiber-reinforced gel where they must depend entirely on the diffusion of oxygen and nutrients through the gel and depend on glucose transporter I and the anaerobic glycolysis machinery to make their ATP [2,37]. They are thus limited in their activities by this restricted fuel supply compared to the osteocytes with their substantial mitochondrial complements and L-C contact with the blood circulation. Nevertheless, chondrocytes still sense and respond to the movements of their joints. So how might an antisocial, oxygen-deprived, articular chondrocyte, with its limited ATP making ability, feel the movement of its joint? One of the ways, maybe the most important one, is with a compression-toggled solitary (primary) cilium. The chondrocyte cilium was first mentioned by Hart in a 1968 abstract [39] and a decade later by Wilsman and colleagues [39–41]. Since then it has been studied largely by C.A. Poole and his colleagues in Auckland New Zealand long before the osteoporosis-driven explosion of interest in bone signaling and now the osteoblast/osteocyte cilium. Chondrocytes are encapsulated in fibrillar-walled chondrons which are embedded in an ICM (interchondronal matrix), a hydroelastic collagenous composite [38]. The chondron is distinguished by being selectively enriched with the minor collagen VI and water-binding proteoglycans (hyaluoronan and aggrecan)
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[38,42]. Because of the water associated with the proteoglycans, the first person to recognize the chondron, Benninghof [43], regarded them as fluid-filled sacs or bladders. In fact when the cartilage and its chondrons are strongly and cyclically compressed as their owner stands up and starts walking the water associated with the chondron proteoglycans is squeezed through the PCC (pericellular capsule) wall into the much less strained ICM (i.e., the PCC has a smaller compression modulus than the ICM; it takes less force to produce a given amount of PCC strain than ICM strain) leaving the chondron with a higher osmotic pressure because of the compression-concentrated proteoglycans and salts. Along with the water squeezed out during each compression cycle, various chondrocyte products including wastes and those needed to maintain the joint are filtered through the PCC meshwork The intrachondronal pressure is prevented by the strong PCC from dissipating and the chondron collapsing. When the compression drops during each cycle, water with chondrocyte nutrients is drawn back into the decompressed chondron because of the high osmotic pressure produced during compression [38]. By straining the PCC’s fibrillar meshwork and pushing the PCC wall closer to the cell surface during the flattening of the chondron during each compression cycle the chondrocyte cilium with meshwork fibrils attached to it will be bent [44,45] (Fig. 2); (see also the chondron cartoons in Fig. 5 of Poole [38]). The upper part of the cilium is decorated with plaques containing α2β1 and α3β1 integrins which are tethered to the pericellular collagen fibers [44,45] (Fig. 2). The bending of the cilium by an encroaching PCC wall and/or the tugging on the integrins by pericellular collagen fibrils in the flatterning chondron will generate a cascade of events ranging from internal Ca2+ release to chondrocyte-appropriate gene activations [46] (Fig. 2) We do not
Fig. 2. Unlike the osteocytes in their osteointernet, articular chondrocytes are not parts of a chondrointernet. Instead they are enclosed in chondron capsules suspended in an avascular collagenous composite. The chondrocyte cilium is decorated in its upper part with α2β1 and α3β1 integrins-containing plaques to which are attached collagen (e.g., collagen VI) meshwork fibrils. When the cartilage is compressed while standing, walking, or running the cilium is bend by the encroaching chondron wall and by being pulled by the collagen fibrils in the compressed chondron. While little is currently known about the signals sent into the chondrocyte by its bending cilium one of the signals is likely an inflow of Ca2+ and the many events this is likely to trigger.
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yet know whether the chondrocyte cilium has PKD1 (PC-1) and TRPP2 (PC-2) as do the kidney cell and osteoblast/osteocyte cilia. But according to Guilak et al. [47] and Yellowley et al. [48,49], fluid flow or deformation of the chondrocytes with a micropipette induces the inflow of Ca2+ through a mechanosensitive channel as well as the IP3-induced release of Ca2+ from internal stores. This hints at the cilia possibly having the full, toggle-activatable PKD1 (PC-1)•TRPP2 (PC-2) signalplex. While this discussion has focused on the response of the solitary (primary) cilium of an articular chondrocyte in its chondron to the strains of cyclical compression, the chondrocyte cilium has a major role in endochondral bone formation, Factors and events in the endochondral growth and development of a long bone such as the femur have been recently reviewed by Kronenberg [50] and very recently by Whitfield [2]. Endochondral bone formation is driven from cartilaginous growth plates at the ends of the bone. The round, moderately sized chondrocytes in the reserve or resting zone at the top of the growth plate nearest the epiphyses are the chondroprogenitor cells. When a progenitor cell is signaled to start a cell cycle and divides, one of its daughters stays in the home niche while the other joins one of several parallel columns or stacks of flat, proliferating, transit-amplifying cells making aggrecan, collagen II, link protein and the gene for CMP (cartilage matrix protein), the specific marker for cells in the pre-hypertrophic zone. Eventually they stop proliferating, turn off their protective anti-apoptosis Bcl-2 protein, and switch on the terminal hypertrophic program which ends in their apoptotic death and the conversion of the cartilage to vascularized bone. Cilium generation in murine growth plate chondrocytes can be prevented by disabling the gene for the Kif3a subunit of the kinesin motor that carries the various structural and functional components up along the ciliary microtubules [51]. This lack of cilia causes dwarfism by causing the misalignment of mitotic prehypertrophic chondrocytes and their daughters along the columns’ longitudinal axes and the severe shortening of the prehypertrophic zone by accelerating the onset of the terminal hypertrophic program [51]. The reason for this striking shortening is due to the absence of Smo (smoothened)•Ptc (patched) complexes that would normally have been located on the solitary cilia [21,52]. When the transitamplifying pre-hypertrophic cells stop proliferating and start terminally hypertrophying, they also start making Ihh which reaches the tops of the pre-hypertrophic columns, binds to the cells’ Ptc receptors which release active Smo from the ciliary Smo•Ptc complexes. The liberated Smo then induces the expression of TGFβ via the Gli transcription factor. The TGF-β in turn stimulates the pre-hypertrophic cells to express and release PTHrP (parathyroid hormone-related peptide) (reviewed in [2,50]). The secreted PTHrP keeps the prehypertrophic cells proliferating and prevents them from initiating the hypertrophic program and the cartilage-bone transition. Thus, the chondrocyte cilium is a major part of the feedback mechanism that determines the structure and size of the pre-hypertrophic zone and thus the length of the adult bone. 5. Conclusion\cilia and the lively skeleton According to the story so far, the solitary (primary) cilium, once believed to be just a relict of early cellular evolution,
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controls, among many other things, endochondral bone formation and the responses of mature bone and cartilage to mechanical strain. The Ihh receptor complex Smo•Ptc on the cilia of transitamplying chondrocytes in developing bone is part of the IhhPTHrP feedback mechanism that determines the length of the prehypertrophic zone and thus the final adult bone length. In adult bones, the cyclical compression produced by various activities sends pulses of fluid through the bones’ extensive L-C steointernet. These pulses toggle the osteocytes’ solitary (primary) cilia which send waves of Ca2+ and Ca2+-mobilizaing IP3 signals through the L-C osteointernet. These ciliary signals keep the osteogenic genes active to maintain an optimal bone mass and strength. Without them bones weaken and become increasingly likely to fracture if re-loaded. Unlike the extensively interconnected osteoblasts, the chondrocytes in the articular cartilage are solitary and individually enclosed in chondron capsules. The cyclic compression of the articular cartilage in the moving skeleton, flattens the chondrons with their enclosed chondrocytes. This bends the cilia which send Ca2+ signals into the cell to keep the genes for cartilage components active to maintain and renew the cartilage. While the solitary (primary) cilium now appears to be a major mechanosensory device in bone and cartilage we still know very little about how it is toggled, the signals it sends and how it relates to the other mechanosensing mechanisms. References [1] R.B. Martin, D.B. Burr, N.A. Sharkey, Skeletal Tissue Mechanics, Springer-Verlag, New York, 1998. [2] J.F. Whitfield, Growing Bone, 2ed.Landes Bioscience, Austin, TX, 2007. [3] J.F. Whitfield, Cancer Lett. 244 (2006) 8. [4] U. Sharma, A.G. Mikos S.C. Cowin, in Mechanosensory Mechanisms in Bone, R.Lanza, R. Langer, J. Vancanti eds., Chapter 61, Elsevier/ Academic Press, San Diego, 2007. [5] L.C. Johnson, Birth Defects Orig. Artic. Ser. 2 (1966) 66. [6] N.X. Chen, K.D. Ryder, F.M. Pavalko, C.H. Turner, D.B. Burr, J. Qiu, R.L. Duncan, Am. J. Physiol., Cell Physiol. 278 (2000) C989. [7] S.W. Donahue, H.J. Donahue, C.R. Jacobs, Am. J. Physiol., Cell Physiol. 281 (2001) C1635. [8] S.W. Donahue, H.J. Donahue, C.R. Jacobs, J. Biomech. 36 (2003) 35. [9] J. Klein-Nulend, P.J. Nijweide, Curr. Osteoporosis Rep. 1 (2003) 5. [10] J. Klein-Nulend, C.M. Semeins, N.E. Ajubi, P.J. Nijweide, E.H. Burger, Biochem. Biophys. Res. Commun. 217 (1995) 640. [11] J. Klein-Nulend, A. van der Plas, C.M. Semeins, N.E. Ajubi, J.A. Frangos, P.J. Neiweide, E.H. Burger, FASEB J 9 (1995) 441. [12] P.A. Janmey, C.A. McCulloch, Annu. Rev. Biomed. Eng. 9 (2007) 1. [13] J.F. Whitfield, J. Cell. Biochem. 89 (2003) 233. [14] K.W. Zimmermann, Arch. Mikrosk. Entwickl.Mech. 52 (1898) 552. [15] E.A. Tonna, N.M. Lampen, J. Gerontol. 27 (1972) 316. [16] H.A. Praetorius, K.R. Spring, J. Membrane Biol. 184 (2001) 71. [17] S.M. Nauli, F.J. Alenghat, Y. Luo, E. Williams, P. Vassilev, X. Li, A.E. Elia, W. Lu, E.M. Brown, S.J. Quinn, D.E. Ingber, J. Zhou, Nat. Genet. 33 (2003) 129. [18] P. Satir, S.V. Christensen, Annu. Rev. Physiol. 69 (2007) 377. [19] V. Chauvet, X. Tian, H. Husson, D.H. Grimm, T. Wang, T. Hieseberger, P. Igarashi, A.M. Bennett, O. Ibraghimov-Beskrovnaya, S. Somlo, M.J. Caplan, J. Clin. Invest. 114 (2004) 1433.
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