Kalirin : novel role in osteocyte function

Abstract

Communication between bone cells is important for the maintenance of bone mass. Although osteocytes are deeply embedded within the mineralized matrix, they are essential for the regulation of osteoblast and osteoclast functions. However, the intracellular proteins that control the morphology and function of osteocytes, and their ability to communicate with other bone cells are still unknown. Kalirin is a novel multi-domain GTP exchange factor (GEF) protein that activates the RhoGTPases. Recently, we found that 14 week old female Kalirin knockout (Kal-KO) mice exhibit a 45% decrease in trabecular bone density and have significantly lower cortical area, perimeter, thickness and polar cross-sectional moment of inertia (-12.6%, -7.2%, -7.6% and -21.9%, respectively) than WT mice. Kalirin was found to be expressed in osteoclasts and osteoblasts but its expression and function in osteocytes is currently unclear. We examined the role of Kalirin on the morphology and function of osteocytes. Primary osteocytes were isolated by sequential collagenase digestions from long bones (femurs and tibias) of 10-week old WT and Kal-KO mice. Immunofluorescent staining revealed Kalirin was localized to the perinuclear region of primary osteocytes and MLO-Y4 cells, and was detected along the cytoplasmic processes of primary osteocytes. We also examined primary osteocytes isolated from the long bones of Kal-KO and WT mice for changes in the length and number of cytoplasmic processes. Kal-KO osteocytes were found to express significantly fewer cytoplasmic processes per cell (3.3±0.21) than WT osteocytes (4.7±0.3). In addition, the cytoplasmic processes of Kal-KO osteocytes were shorter (79.5±4.6 µm) than those observed for WT osteocytes (85.4±3.6 µm) (p <0.01). Quantitative PCR revealed the expression of mRNA for the three major Kalirin isoforms (Kal-7, Kal-9, Kal-12) in primary osteocytes and in MLO-Y4 cells. Moreover, the mRNA levels of osteoprotegerin (OPG) and SOST, which are important for controlling osteoclast differentiation and Wnt signaling leading to bone formation, respectively, were reduced in Kal-KO osteocytes. Next, the role of Kalirin in osteocyte morphology and function was further examined. Treatment of MLO-Y4 cells for 5 days with nerve growth factor, which is known to activate Kalirin in neurons, or over-expression of the Ser-Thr kinase domain of Kal-12, promoted cytoplasmic process elongation and upregulated phosphorylated ERK and RhoA levels. Together, these results suggest that Kalirin controls osteocyte morphology and function in part by regulating cytoskeletal remodeling and the activity of ERK and RhoA. Furthermore, Kalirin may control the bone remodeling cycle by regulating osteocyte signaling to osteoclasts and osteoblasts.