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DLK is a key regulator of axon regeneration and degeneration in response to neuronal injury. To understand the molecular mechanisms controlling the DLK function, we performed yeast two-hybrid screening analysis and identified FKBPL as a DLK-binding protein that bound to the kinase domain and inhibited the kinase enzymatic activity of DLK. FKBPL regulated DLK stability through ubiquitin-dependent DLK degradation. We tested other members in the FKBP protein family and found that FKBP8 also induced DLK degradation as FKBPL did. We found that Lysine 271 residue in the kinase domain of DLK was a major site of ubiquitination and SUMO3-conjugation and responsible for FKBP8-mediated degradation. In vivo overexpression of FKBP8 delayed progression of axon degeneration and neuronal death following axotomy in sciatic and optic nerves, respectively, although axon regeneration efficiency was not enhanced. This research identified FKBPL and FKBP8 as new DLK-interacting proteins that regulated DLK stability by MG-132 or bafilomycin A1-sensitive protein degradation.

Bone is a dynamic tissue under constant remodeling in response to various signals including mechanical loading. A lack of proper mechanical loading induces disuse osteoporosis that reduces bone mass and structural integrity. The β-catenin signaling together with a network of GTPases is known to play a primary role in load-driven bone formation, but little is known about potential interactions of β-catenin signaling and GTPases in bone loss. In this study, we addressed a question: Does unloading suppress an activation level of RhoA GTPase and β-catenin signaling in osteoblasts? If yes, what is the role of RhoA GTPase and actin filaments in osteoblasts in regulating β-catenin signaling? Using a fluorescence resonance energy transfer (FRET) technique with a biosensor for RhoA together with a fluorescent T cell factor/lymphoid enhancer factor (TCF/LEF) reporter, we examined the effects of clinostat-driven simulated unloading. The results revealed that both RhoA activity and TCF/LEF activity were downregulated by unloading. Reduction in RhoA activity was correlated to a decrease in cytoskeletal organization of actin filaments. Inhibition of β-catenin signaling blocked unloading-induced RhoA suppression, and dominant negative RhoA inhibited TCF/LEF suppression. On the other hand, a constitutively active RhoA enhanced unloading-induced reduction of TCF/LEF activity. The TCF/LEF suppression by unloading was enhanced by co-culture with osteocytes, but it was independent on the organization of actin filaments, myosin II activity, or a myosin light chain kinase. Collectively, the results suggest that β-catenin signaling is required for unloading-driven regulation of RhoA, and RhoA, but not actin cytoskeleton or intracellular tension, mediates the responsiveness of β-catenin signaling to unloading.

Chondrocytes are the only cell type present in the articular cartilage and their response to mechanical stimuli influences the maintenance and remodeling of the cartilage. Numerous studies have shown that the balance between anabolic and catabolic responses of the chondrocytes to mechanical loading is dependent on the loading intensities (reviewed in ref. [1]). Moderate, physiological loading, for instance, increases synthetic activity of the extracellular matrix (ECM) such as collagen type II, aggrecan, and proteoglycan, while decreasing the catabolic activity of degradative enzymes such as matrix metalloproteinases (MMPs) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) [2,3]. In contrast to moderate loading, static or high-intensity loading has been shown to degrade the cartilage resulting from inhibition of matrix synthesis and up-regulation of catabolic activities [3,4]. Therefore, the importance of these load-dependent signaling pathways involved in the maintenance and remodeling of the cartilage is widely accepted. However, the underlying mechanisms as to how varying magnitudes of mechanical stimuli trigger differential signaling activities that consequently lead to selective gene expression are not clear. FAK and Src are considered to be the main mechanotransduction signaling proteins at the cell-ECM adhesion sites and their activities influence various structural and signaling changes within the cell, including cytoskeletal organization, migration, proliferation, differentiation, and survival [5]. Accumulating evidence has shown that Src and FAK play crucial roles in regulating cartilage maintenance and degeneration and their activation stimulates matrix catabolic genes and activity [6,7]. Rho family GTPases such as RhoA and Rac1 play critical roles in fundamental processes including morphogenesis, polarity, movement, and cell division [8]. They also contribute to cartilage development and degradation [9,10]. Despite these studies, much remains to be elucidated on how load-induced Src and FAK participate in chondrocyte functions, and how their interactions are linked and regulated in connection to the activities of RhoA and Rac1 under different loading conditions. In this study, we use fluorescence resonance energy transfer (FRET)-based biosensors to monitor activities of Src and FAK as well as individual GTPases and evaluate the potential linkage of a network of these signaling molecules under different loading conditions.Copyright © 2013 by ASME

Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate that both Rac1 and Cdc42 GTPases are critical regulators in shear stress-driven β-catenin signaling in osteoblasts.

Osteoblasts play a pivotal role in load-driven bone formation by activating Wnt signaling through a signal from osteocytes as a mechanosensor. Osteoblasts are also sensitive to mechanical stimulation, but the role of RhoA, a small GTPase involved in the regulation of cytoskeleton adhesion complexes, in mechanotransduction of osteoblasts is not completely understood. Using MC3T3-E1 osteoblast-like cells under 1 hr flow treatment at 10 dyn/cm(2), we examined a hypothesis that RhoA signaling mediates the cellular responses to flow-induced shear stress. To test the hypothesis, we conducted genome-wide pathway analysis and evaluated the role of RhoA in molecular signaling. Activity of RhoA was determined with a RhoA biosensor, which determined the activation state of RhoA based on a fluorescence resonance energy transfer between CFP and YFP fluorophores. A pathway analysis indicated that flow treatment activated phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling as well as a circadian regulatory pathway. Western blot analysis revealed that in response to flow treatment phosphorylation of Akt in PI3K signaling and phosphorylation of p38 and ERK1/2 in MAPK signaling were induced. FRET measurement showed that RhoA was activated by flow treatment, and an inhibitor to a Rho kinase significantly reduced flow-induced phosphorylation of p38, ERK1/2, and Akt as well as flow-driven elevation of the mRNA levels of osteopontin and cyclooxygenase-2. Collectively, the result demonstrates that in response to 1 hr flow treatment to MC3T3-E1 cells at 10 dyn/cm(2), RhoA plays a critical role in activating PI3K and MAPK signaling as well as modulating the circadian regulatory pathway.