Sara Temiyasathit

Osteocyte Mechanobiology and Pericellular Mechanics

An impressive range of tissues and cells are regulated by mechanical loading, and this regulation is central to disease processes such as osteoporosis, atherosclerosis, and osteoarthritis. However, other than a small number of specialized excitable cells involved in hearing and touch, cellular mechanosensing mechanisms are generally quite poorly understood. A lack of mechanistic understanding of these processes is one of the primary foci of the nascent field of mechanobiology, which, as a consequence, enjoys enormous potential to make critical new insights into both physiological function and etiology of disease. In this review we outline the process in bone by tracing mechanical effects from the organ level to the cellular and molecular levels and by integrating the biological response from molecule to organ. A case is made that a fundamental roadblock to advances in mechanobiology is the dearth of information in the area of pericellular mechanics.

Primary cilium-dependent mechanosensing is mediated by adenylyl cyclase 6 and cyclic AMP in bone cells

Primary cilia are chemosensing and mechanosensing organelles that regulate remarkably diverse processes in a variety of cells. We previously showed that primary cilia play a role in mediating mechanosensing in bone cells through an unknown mechanism that does not involve extracellular Ca2+‐dependent intracellular Ca2+ release, which has been implicated in all other cells that transduce mechanical signals via the cilium. Here, we identify a molecular mechanism linking primary cilia and bone cell mechanotransduction that involves adenylyl cyclase 6 (AC6) and cAMP. Intracellular cAMP was quantified in MLO‐Y4 cells exposed to dynamic flow, and AC6 and primary cilia were inhibited using RNA interference. When exposed to flow, cells rapidly (<2 min) and transiently decreased cAMP production in a primary cilium‐dependent manner. RT‐PCR revealed differential expression of the membrane‐bound isoforms of adenylyl cyclase, while immunostaining revealed one, AC6, preferentially localized to the cilium. Further studies showed that decreases in cAMP in response to flow were dependent on AC6 and Gd3+‐sensitive channels but not intracellular Ca2+ release and that this response mediated flow‐induced COX‐2 gene expression. The signaling events identified provide important details of a novel early mechanosensing mechanism in bone and advances our understanding of how signal transduction occurs at the primary cilium.—Kwon R. Y., Temiyasathit, S., Tummala, P., Quah, C. C., Jacobs, C. R. Primary cilium‐dependent mechanosensing is mediated by adenylyl cyclase 6 and cyclic AMP in bone cells. FASEB J. 24, 2859–2868 (2010). www.fasebj.org

Osteocyte primary cilium and its role in bone mechanotransduction

Bone is a dynamic tissue that adapts to its local loading environment. Mechanotransduction, the process by which cells convert mechanical forces into biochemical signals, is important for maintaining bone health and homeostasis. It is less clear, however, what the cellular mechanosensor(s) are that sense and initiate these signaling cascades. Primary cilia are solitary rigid structures that extend from the cell body into the extracellular space and as a consequence are prime candidates for mechanosensing in bone. Primary cilia have been shown to be critical in development and have been implicated in mechanosensing in other tissue types, including liver and kidney. In this review we discuss the potential for primary cilia to play an important role in bone mechanotransduction and possible avenues for future study.

Mechanosensing by the Primary Cilium: Deletion of Kif3A Reduces Bone Formation Due to Loading

Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Colα1(I) 2.3-Cre;Kif3afl/fl mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Colα1(I) 2.3-Cre;Kif3afl/fl and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Colα1(I) 2.3-Cre;Kif3afl/fl mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.

Primary cilia act as mechanosensors during bone healing around an implant

The primary cilium is an organelle that senses cues in a cells local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

CXCR4 antagonism attenuates load-induced periosteal bone formation in mice.

Mechanical loading is a key anabolic regulator of bone mass. Stromal cell‐derived factor‐1 (SDF‐1) is a stem cell homing factor that is important in hematopoiesis, angiogenesis, and fracture healing, though its involvement in skeletal mechanoadaptation is virtually unknown. The objective of this study was to characterize skeletal expression patterns of SDF‐1 and CXCR4, the receptor for SDF‐1, and to determine the role of SDF‐1 signaling in load‐induced periosteal bone formation. Sixteen‐week‐old C57BL/6 mice were treated with PBS or AMD3100, an antagonist against CXCR4, and exposed to in vivo ulnar loading (2.8 N peak‐to‐peak, 2 Hz, 120 cycles). SDF‐1 was expressed in cortical and trabecular osteocytes and marrow cells, and CXCR4 was primarily expressed in marrow cells. SDF‐1 and CXCR4 expression was enhanced in response to mechanical stimulation. The CXCR4 receptor antagonist AMD3100 significantly attenuated load‐induced bone formation and led to smaller adaptive changes in cortical geometric properties as determined by histomorphometric analysis. Our data suggest that SDF‐1/CXCR4 signaling plays a critical role in skeletal mechanoadaptation, and may represent a unique therapeutic target for prevention and treatment of age‐related and disuse bone loss.

Adenylyl Cyclase 6 Mediates Primary Cilia-Dependent Changes in Cyclic Adenosine Monophosphate in Response to Dynamic Fluid Flow

It is well accepted that fluid flow is an important mechanical signal in regulating bone structure and function. Primary cilia, which are solitary, microtubule-based organelles that extend from the centrosome into extracellular space in many cell types, have been shown to mediate fluid flow-induced osteogenic responses in MLO-Y4 osteocyte-like cells [1], however, primary cilia did not mediate increases in intracellular Ca2+ concentration [1]. Recently, we identified cAMP as a novel early signaling molecule in primary cilia-dependent mechanotransduction of fluid flow in osteocytes. Specifically, we show that MLO-Y4 osteocyte-like cells respond to oscillatory flow with a rapid decrease in intracellular levels of cAMP that is dependent on the primary cilium [2]. Adenylyl cyclase 6 (AC6) is an enzyme responsible for the synthesis of cAMP from ATP. We found that AC 6 localizes to the primary cilium of bone cells (Fig. 1). In this study, our goal was to determine whether AC6 mediates the primary cilia-dependent, flow-induced decrease in cAMP.Copyright

Primary Cilia Mediate Intracellular cAMP Responses in Bone Cells Exposed to Dynamic Fluid Flow

It is well accepted that fluid flow is an important mechanical signal in regulating bone structure and function. Primary cilia, which are non-motile, microtubule based organelles that extend from the centrosome and project into extracellular space in many cell types, have recently been shown to mediate fluid flow-induced osteogenic responses in MLO-Y4 osteocyte-like cells [1]. However, primary cilia did not mediate increases in intracellular Ca2+ concentration, and the second messenger system(s) involved in primary cilia-mediated mechanosensing has yet to be elucidated. In this study, our goals were to (1) determine whether exposing bone cells to oscillatory fluid flow modulates intracellular levels of cyclic adenosine monophosphate (cAMP), another ubiquitous second messenger molecule, and (2) investigate whether this modulation may be mediated by primary cilia.Copyright